CN101658672A - Combination therapy with class iii anti-cea monoclonal antibodies and therapeutic agents - Google Patents

Abstract

The invention relates to a combination therapy with class III anti-CEA monoclonal antibodies and therapeutic agents. The present invention provides a composition comprising naked humanized, chimeric and human Class III anti-CEA monoclonal antibody and a therapeutic agent, which is useful for the treatment of CEA expressing cancers and other diseases, and methods of treatment using this composition.

Description

Carry out therapeutic alliance with anti-CEA monoclonal antibody of III class and therapeutic agent

The application is to be the Chinese patent application 02830006.8 on October 11st, 2002 dividing an application of " carrying out therapeutic alliance with anti-CEA monoclonal antibody of III class and therapeutic agent " applying date.

Technical field

The present invention relates to treatment and express the carcinoembryonic antigen (" cancer of CEA "), particularly medullary thyroid carcinoma (MTC), non-medullary thyroid carcinoma (non-MTC), colorectal carcinoma, hepatocarcinoma, gastric cancer, pulmonary carcinoma, breast carcinoma and other are expressed the method for cancer of CEA, wherein said treatment is by giving a kind of immunoreagent that comprises the antibody of a kind of and at least a other therapeutic agent combination, described other therapeutic agent such as chemotherapeutant, radiological agent, immunomodifier, immune conjugate or its combination.The invention further relates to the pharmaceutical composition that comprises immunoreagent and at least a therapeutic agent that exists with non-coupling form.Especially, the present invention relates to by before giving therapeutic agent, simultaneously or give the anticancer embryonal antigen of III class (anti-" CEA ") monoclonal antibody (" MAb ") afterwards, particularly have binding affinity characteristic and corresponding Mus III class anti--the specific MAb of CEA MAb, the MAbs humanized, chimeric or the people that has the antigenicity and the effector characteristic of various human antibody more in particular treats the method for cancer of expressing CEA.Useful especially MAbs is humanized MAbs in the method for treatment, wherein will resist complementary determining region (" the CDRs ") grafting of the Mus MAb of CEA to go into the framework region of people's antibody.

Background technology

CEA is a kind of carcinoembryonic antigen that reaches at many kinds of epithelium cancer invading the exterior usually, described epithelium cancer is that those great majority appear at the colon cancer (Goldenberg etc. in breast, lung, pancreas, thyroid (marrow type) and ovary in addition, J Natl.CancerInst.57:11-22 (1976), Shively, Deng, Crit.Rev.Oncol.Hematol.2:355-399 (1985)).CEA is considered to the tumor specific antigen (Gold etc., JExper.Med., 122:467 (1965)) of colorectal carcinoma at the beginning.But, found afterwards that it was present in multiple cancer, benign tumor and the pathological tissue, and (Shively etc., Crit.Rev.Oncol.Hemato., 2:355 (1985) in normal people's colon; Von Kleist etc., Proc.Natl.Acad.Sci.U.S.A., 69:2492 (1972)).CEA has shown that this has then pointed out the effect of CEA in tumorigenic various aspects by homotype and the special-shaped adjusting cell that interacts-cell absorption.

Being limited to thyroid medullary thyroid carcinoma (MTC) can dissect and smelting is healed by thyroid complete resection and maincenter lymph node.But, disease these patients about 50% in the recurrence.In addition, the prognosis of suffering from the patient that unresectable disease or far-end shift is no good, less than 30% patient survive 10 years (Rossi etc., Amer.J Surgery, 139:554 (1980); Samaan etc., J Clin.Endocrinol.Metab., 67:801 (1988); Schroder etc., Cancer, 61:806 (1988).These patients are left few therapy apparatus meeting (Principles and Practice of Oncology, DeVita, Hellman and Rosenberg (editor), New York: JB Lippincott Co.1333-1435 (1989); Cance etc., Current Problems Surgery, 22:1 (1985)).Chemotherapy is worth little and radiotherapy only can be used for controlling (the Cance etc. of local disease; Tubiana etc., Cancer, 55:2062 (1985)).Thereby, need new form of therapy to control this disease.

Utilize targeting antibodies directly to tumor sites delivering therapeutic agents and diagnostic agent a kind of treatment of cancer is comprised with the diagnosis useful method.In the past ten years, many tumor specific antibodies and antibody fragment have been developed, and antibody coupling is attached on therapeutic agent as medicine, toxin, radionuclide, the immunomodulator and other medicament as cytokine, and with the conjugate of target tumor method to patient's administration.But, produce circulative human anti-mouse antibody (HAMAs) with compound with the medicine of mouse monoclonal antibody (it is the most frequently used targeting antibodies for the people) or the patient of radio nuclide therapy, produce general III type anaphylactic reaction immediately sometimes the antibody position of conjugate.But made still less that by the immunogenicity that makes these murine antibody by many diverse ways these problems have minimized, described method comprises by the chemical modification targeting antibodies, as by with polyethylene glycol conjugation (Pegylation) to targeting antibodies, or be tested and appraised the antigenic site in the antibody and subsequently it removed and produce antibody humanized, chimeric or the people; For example, Fab ', F (ab) ₂With other antibody fragment that replaces complete IgG to use.In addition, HAMA is removed the trial that reduces the HAMA side effect from blood by plasma removing.Also use immunorepressive technology to improve the side effect of exogenous antibodies, described exogenous antibodies is enough to allow repeatedly to treat with the targeting medicament

Although have these treatments progressive, the needs that provide more effective treatment to express the method for cancer of CEA still be provided.The invention provides and utilize anti-CEA antibody, as at U.S. Patent number 5,874,540 and Hansen etc., Cancer, the III class anti-CEAMAb Mus MN-14MAb that is introduced among the 71:3478 (1993) and also be at U.S. Patent number 5,874, chimeric and the humanized MN-14 of the anti-CEA MAb of III class that is introduced in 540, and at the U.S. Patent number 4,818 of Primus etc., defined NP-4 in 709 is incorporated herein by reference at this full content with all documents.Preferably, the anti-CEA MAb of III class is humanized, and with a kind of therapeutic agent, the use that combines of particularly a kind of chemotherapeutics is effectively treated the cancer of expressing CEA to produce with minimum toxicity.In addition, the administration respectively of these the two kinds of components versatility and the motility that enhanced results are provided and have been applicable to the individualized treatment method.

Summary of the invention

Claimed among the present invention is the compositions and the method for treatment medullary thyroid carcinoma and non-medullary thyroid carcinoma.

First embodiment of the present invention is a kind of compositions that comprises the anti-CEA monoclonal antibody of at least a III class (MAb) or its fragment and at least a therapeutic agent at least.Preferably, antibody fragment is selected from F (ab ') ₂, Fab ', Fab, Fv and scFv.Also will be preferably, the anti-CEA MAb of III class or its fragment are chimeric MAb, and wherein chimeric MAb keeps the anti-CEA binding specificity of III class of the anti-CEA MAb of Mus III class substantially.Still will be preferably, the anti-CEA MAb of III class or its fragment are people MAb completely, and the wherein said MAb of people completely keeps the anti-CEA binding specificity of III class of the anti-CEA MAb of Mus III class substantially.

In one embodiment of the invention, the anti-CEA monoclonal antibody of III class or its fragment are preferably MN-14 antibody or its fragment.More preferably, MN-14 monoclonal antibody or its fragment comprise the complementary determining region (CDRs) of Mus MN-14 monoclonal antibody, and wherein the CDRs of the variable region of light chain of MN-14 antibody comprises the CDR1 that contains aminoacid sequence KASQDVGTSVA; The CDR2 that contains aminoacid sequence WTSTRHT; With the CDR3 that contains aminoacid sequence QQYSLYRS; And the CDRs of the variable region of heavy chain of the anti-CEA antibody of III class comprises the CDR1 that contains TYWMS; The CDR2 that contains EIHPDSSTINYAPSLKD; With the CDR3 that contains LYFGFPWFAY.Also will be preferably, MN-14 monoclonal antibody and CEA react and do not react with normal cross reacting antigen (NCA) and meconium antigen (MA).Most preferably, MN-14 monoclonal antibody or its fragment are humanized, chimeric or people MN-14 antibody or its fragment completely.

In a preferred embodiment, the framework region of the light chain of humanization MN-14 antibody and variable region of heavy chain (FRs) comprises the aminoacid of at least one replacement from the corresponding FRs of Mus MN-14 monoclonal antibody.Particularly, humanization MN-14 antibody or its fragment preferably comprise at least one aminoacid from the corresponding FR of Mus MN-14 antibody, and described aminoacid is selected from amino acid residue 24 (A), 28 (D), 30 (T), 48 (1), 49 (G), 74 (A) and 94 (S) of the Mus variable region of heavy chain (KLHuVhAI GA) of Figure 14 A-C.Similarly, humanized MN-14 antibody or its fragment can also comprise at least one aminoacid from the corresponding FR of described Mus MN-14 variable region of light chain.Still will be preferably, humanized MN-14 antibody or its fragment comprise variable region of light chain as shown in FIG. 13A, and the variable region of heavy chain that is marked as KLHuVhAIGA shown in Figure 14 A-C, described variable region of light chain is by MN14VK that forms from the combination of the sequence of two Vks of these antibody and the intermediate sequence between the REIVK.

In first embodiment of the present invention, therapeutic agent is selected from naked antibody, cytotoxic agent, medicine, radionuclide, immunomodulator, photosensitive therapeutic agent, immune conjugate, hormone or its combination, optional being formulated in a kind of pharmaceutically useful carrier.Here also requiring therapeutic agent is not dacarbazine (DTIC).

Second embodiment of the present invention described a kind of method for the treatment of marrow sample and non-marrow sample medullary thyroid carcinoma, comprise simultaneously or treat to the experimenter in order the anti-CEA monoclonal antibody of III class or its fragment and at least a therapeutic agent of effective dose, and optional being formulated in a kind of pharmaceutically useful carrier.Preferably, antibody fragment is selected from F (ab ') ₂, Fab ', Fab, Fv and scFv.Also will be preferably, the anti-CEA monoclonal antibody of III class or its fragment are humanized, wherein said humanized monoclonal antibody has kept the anti-CEA binding specificity of III class of the anti-CEA monoclonal antibody of Mus III class substantially.Considered that also the anti-CEA monoclonal antibody of III class or its fragment are chimeric monoclonal antibody, and wherein said mosaic monoclonal antibody has kept the anti-CEA binding specificity of III class of the anti-CEA monoclonal antibody of Mus III class substantially.

In a preferred embodiment, the anti-CEA monoclonal antibody of III class or its fragment are MN-14 antibody or its fragment.Preferably, MN-14 antibody or its fragment comprise the complementary determining region (CDRs) of Mus MN-14 monoclonal antibody, and the CDRs of the variable region of light chain of wherein said MN-14 monoclonal antibody comprises the CDR1 that contains aminoacid sequence KASQDVGTSVA; The CDR2 that contains aminoacid sequence WTSTRHT; With the CDR3 that contains aminoacid sequence QQYSLYRS; And the CDRs of the variable region of heavy chain of the anti-CEA antibody of III class comprises the CDR1 that contains TYWMS; The CDR2 that contains EIHPDSSTINYAPSLKD; With the CDR3 that contains LYFGFPWFAY.Also will be preferably, the MN-14 monoclonal antibody is humanized, chimeric or people MN-14 antibody completely, does not react with the CEA reaction and with normal cross reacting antigen (NCA) and meconium antigen.Also will be preferably, the dosage of MN-14 antibody or its fragment 100-600 milligram/agent/injection carries out administration.The most preferably, MN-14 antibody or its fragment are carried out administration with the dosage of 300-400 milligram/agent/injection.

In the method for the invention, the framework region (FRs) of described humanization MN-14 antibody or its segmental light chain and variable region of heavy chain comprises the aminoacid of at least one replacement from the corresponding FRs of Mus MN-14 monoclonal antibody.More preferably, the amino acid residue 24,28,30,48,49,74 and 94 that comprises at least one Mus variable region of heavy chain that is selected from Figure 14 A-C as mentioned above from amino acid whose humanization MN-14 antibody or its fragment of the described corresponding FR of described Mus MN-14 antibody.Also will be preferably, humanized MN-14 antibody or its fragment comprise at least one aminoacid from the corresponding FR of described Mus MN-14 variable region of light chain.The most preferably, humanized MN-14 antibody or its fragment comprise the variable region of light chain shown in Figure 13 A (intermediate sequence) or Figure 22 A (hMN-14) or Figure 16 A, and be marked as shown in Figure 14 A-C KLHuVhAIGA's or Figure 15 B (hMN-14) or Figure 16 B shown in variable region of heavy chain.

Method of the present invention also comprises the therapeutic agent that is selected from down group: with EGP-1, EGP-2 (for example, 17-1A), humanized, chimeric, the people's or Mus monoclonal antibody or its fragment of MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, EGP-2, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn, Thomson-Friedenreich antigen, neoplasm necrosis antigen, VEGF or other tumor vessel generation antigen, Ga 733 or its composite reaction.Similarly, these methods can comprise simultaneously or treat to the experimenter in order second humanized, chimeric, people or mouse monoclonal antibody or its fragment of effective dose, and described monoclonal antibody or its fragment are selected from II class or the anti-CEA monoclonal antibody of III class or its fragment.Preferably, second antibody or its fragment both can be naked also can being coupled on a kind of therapeutic agent.

In a preferred embodiment of methods described herein, therapeutic agent is selected from immune conjugate, hormone or its combination of naked antibody, cytotoxic agent, medicine, radionuclide, immunomodulator, photosensitive therapeutic agent, CEA or non-CEA antibody, optional being formulated in a kind of pharmaceutically useful carrier.Here also requiring therapeutic agent is not dacarbazine (DTIC).

Preferably, therapeutic agent is a kind of cytotoxic agent that is selected from medicine or toxin.For example, consider that medicine has the pharmacological characteristics that is selected from antimitotic agent, alkylating agent, antimetabolite, anti-angiogenic agent, apoptosis agent, alkaloid, COX-2 and antibiotic and combination thereof.Preferably, medicine is selected from chlormethine, aziridine derivative, alkyl sulfonic ester, nitroso ureas, triazenes, folacin, anthracycline, taxane, cox 2 inhibitor, pyrimidine analogue, purine analogue, antimetabolite, antibiotic, enzyme, epipodophyllotoxin, iridium-platinum complex, vinca alkaloids, replacement urea, methylhydrazine derivant, adrenal cortex inhibitor, antagonist, endostatin, paclitaxel, camptothecine, amycin and their analog, and combination.

When therapeutic agent was toxin microorganism, plant or animal, this medicament can be selected from Ricin, abrin, alpha toxin, ZAOCAO toxin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcal enterotoxin A, Radix Phytolaccae antivirin, gelonin (gelonin), diphtheria toxin, diphtherotoxin, Pseudomonas exotoxin and pseudomonas endotoxin.

Consider also that in the method for the invention therapeutic agent is the immunomodulator that is selected from cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), stem cell factor, erythropoietin, thrombopoietin and combination thereof.Preferably, lymphotoxin is tumor necrosis factor (TNF), described Hemopoietic factor is interleukin (IL), described colony stimulating factor is granulocyte colony-stimulating factor (G-CSF) or granulocyte macrophage colony stimulating factor (GM-CSF), plain interferon is interferon-ALPHA, β or γ, and described stem cell factor is appointed as " the S1 factor ".Also will be preferably, immunomodulator comprises I L-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, IFN-, TNF-α or its combination.Also will be preferably, therapeutic agent be a kind of for chromogen or dyestuff photosensitive therapeutic agent or a kind ofly be the alkylating agent of dacarbazine.

Also will be preferably, therapeutic agent is for having 20-10, the radionuclide of the energy of 000keV.Preferably, this radionuclide is selected from ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁸Y, ²²⁵Ac, ¹⁷⁷Lu, ¹⁸⁸Re, ¹⁸⁶Re and combination thereof.

Description of drawings

Fig. 1. separately with hMN-14, the chart of comparison of tumor size with DTIC or after separately with hMN-14 and DTIC therapeutic alliance.Figure 1A shows with 25 and 100 μ g/ agent separately or with the DTI C of 250 μ g hMN-14 antibody administrations.Figure 1B shows with 50 and 75 μ g/ agent separately or with the DTIC of 100 μ g hMN-14 antibody administrations.

Fig. 2. with ¹³¹I and ⁹⁰Y-MN-14 carries out radioimmunotherapy (RAIT) chart of comparison of tumor size afterwards.

Fig. 3. more several chemotherapeutics in suffering from the mice of TT to the chart of tumor size therapeutic effect.Wherein: suffer from the mice amycin of TT, 20mg/ m ²0,1 and 2 day (70 μ g/ agent) (zero); DTIC, 30mg/ m ²0,1 and 2 day (1.08mg/ agent) (); Amycin and DTIC be (●) as above; The ring phosphonic amide, 600mg/m2; 0 day (2.16mg/ agent) (△); Vincristine, 4.2 μ g/ agent; 0 day (*); All four kinds of medicines (amycin, DTIC, cyclophosphamide and vincristine) more than regard to every kind of described dosage (■); Or handle (◆).Each group is made up of the nude mice that 6-9 only suffers from the TT tumor of foundation.Mean tumour volume during treatment is 0.51 ± 0.33cm ³Point: average tumor size.Error bars: standard deviation only shows so that clear on symbol.

Fig. 4. relatively RAIT and ⁹⁰The combination of the therapeutic alliance of the anti-CEA monoclonal antibody MN-14 of Y labelling and 4 kinds of medicines that began in 24 hours RAIT after is for the chart of the therapeutic effect of tumor size.Wherein: the animal that suffers from tumor be untreated (◆); Give 4 kinds of pharmaceutical admixtures described in Fig. 1, carried out administration (■) at the 1st, 2 and 3 day; Gave 52.5 μ Ci's at 0 day ⁹⁰Y-NH-14 gave 4 pharmacotherapys (▲) subsequently at the 1st, 2 and 3 day; Gave 52.5 μ Ci's at 0 day ⁹⁰Y-NH-14 (Δ); Or gave 105 μ Ci's at 0 day ⁹⁰Y-NH-14 (zero).For untreated fish group N=5, n=9-10 in processed group.Mean tumour volume during treatment is 0.28 ± 0.12cm ³Point: average tumor size.Error bars: standard deviation only shows so that clear on symbol.

Fig. 5. relatively RAIT adds that DTIC and RAIT add the chart of amycin and the effect of DTIC in suffering from the TT mice.Wherein: the mice that suffers from TT is handled (◆); Gave 105 μ Ci's at 0 day ⁹⁰Y-NH-14 (zero); Gave 105 μ Ci's at 0 day ⁹⁰Y-NH-14, subsequently at the 1st, 2 and 3 day with 50% full dosed administration amycin and DTIC (▲); Gave 52.5 μ Ci's at 0 day ⁹⁰Y-NH-14, subsequently at the 1st, 2 and 3 day with 75% full dosed administration DTIC (*); Or at the 1st, 2 and 3 day with 300mg/m ²The DTIC of the full dosage of administration (1.08mg/ agent) ().For untreated fish group N=5, n=9-10 in processed group.Mean tumour volume during treatment is 0.39 ± 0.20cm ³Point: average tumor size.Error bars: standard deviation only shows so that clear on symbol.

Fig. 6. compare the chart of the effect of naked hMN-14 therapeutic scheme in suffering from the xenotransplantation mice of TT.Animal is given the subcutaneous injection of TT cell, and is left and does not carry out treating (A) or give intravenous injection 0.5mg hMN-14 at the 1st day (B) or the 11st day slower (C).Lines in A, B and C picture are represented the gross tumor volume of individual animals.The meansigma methods of each treatment group is shown in the D picture.Error bars is represented the standard error of meansigma methods, and only is shown in a direction so that clear.◆, untreated; handled on the 1st day; △ handled on the 11st day.

Fig. 7. the more humanized or effect of Mus MN-14 antibody in the treatment medullary thyroid carcinoma.Give animal subcutaneous injection TT cell, and stay do not carry out treating or after gave intravenous injection MAb (0.5mg) on the 1st day.The meansigma methods that wherein shows each treatment group.Error bars is represented the standard error of meansigma methods and only is shown in a direction so that clear.◆, untreated; , hMN-14; △, Mus MN-14; *, hLL2.

Fig. 8. the effect of more different hMN-14 dosage in the treatment medullary thyroid carcinoma.After subcutaneous injection TT cell the 1st day gives the hMN-14 dosage that intravenous injection increases gradually.The meansigma methods that shows each treatment group.◆, untreated; ●, 0.125mg; Zero, 0.25mg; ■, 0.50mg; *, 1.0mg; ▲, 2.0mg.Error bars is represented the standard error of meansigma methods, and only shows so that clear for untreated fish group and the group of having accepted 0.50mg hMN-14.

Fig. 9. the chart of the effect of relatively different treatment times in suffering from the nude mice of TT.1 day (●), 3 days (▲) or 7 days (■) after subcutaneous injection TT cell give the hMN-14 of animal intravenous injection 0.25mg,, or stay and do not carry out handling (◆).The meansigma methods that shows each treatment group.Error bars is represented the standard error of meansigma methods, and only is shown in a direction so that clear.

Figure 10. relatively add the chart that DTIC, independent DTIC, independent hMN-14 treatment suffer from the nude mice of TT and do not treat mice with hMN-14.Gave animal peritoneal injection hMN-14 at the 2nd, 3,4,5,7,8,9,10 and 11,15 and 22 day with 100 μ g/ agent, injection in per subsequently 7 days is (zero) once; DTIC (▲) in administration in the 2nd, 3 and 4 day 75 μ g/ agent; The combination of these hMN-14 and DTIC therapy (△); Or do not handle (◆).The meansigma methods that shows each treatment group.10 animal/groups.

Figure 11. Figure 11 A and 11B show Mus MN-14 variable region heavy chain (VH) total DNA sequence and by the aminoacid sequence of this dna sequence encoding.CDRs is drawn together in frame.

Figure 12. Figure 12 A and 12B show Mus MN-14 variable region light chain (VK) total DNA sequence and by the aminoacid sequence of this dna sequence encoding.CDRs is drawn together in frame.

Figure 13. Figure 13 A and 13B show the variable region of Mus MN-14 and the comparison in people variable region NEWM VH and REI VK (Figure 13 A) and people KOL VH district (Figure 13 B).CDRs draws together in the frame, the Mus VH FRs that mixes humanization VH with them according to Kabat etc., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, U.S.Government PrintingOffice, Washington, D.C., labelling is carried out in the position of 1987 number system.The Mus residue that is included in outside the CDRs among the KLHuVH is indicated with a filled circles.

Figure 14. Figure 14 A-14C is presented at the comparison of the aminoacid sequence between Mus and the humanization MN-14VH f framework residue (FR).Only show the people FR residue that is different from mice.The CDRs of NEWM and KOL does not show yet.Give prominence to runic in the zone of the aminoacid replacement in each FRs, and the position of substitution is indicated according to the numbering system of Kaba t etc.3 CDR are drawn together in the frame.

Figure 15. people, Mus and the humanization sequence that Figure 15 A and Figure 15 B show the Vk of people REI and KOL antibody and VH district respectively with the comparison of Mus and humanization MN-14.The human sequence of REIVk among Figure 15 A is compared with Mus and humanization MN-14Vk sequence.The sequence that the filled circles indication is kept by people REI Vk sequence.CDRs is drawn together in the frame.The human sequence of KOL VH among Figure 15 B is compared with Mus and humanization MN-14VH sequence.The sequence that the filled circles indication is kept by people REI Vk sequence.CDRs is drawn together in the frame.

Figure 16. Figure 16 A and 16B show Vk, variable light chain and VH, the variable heavy chain sequence of hMN-14, and described hMN-14 is the anti-CEA antibody of a kind of humanized III class.The CDR district shows with runic and underscore.Amino acid residue and nucleotide number in order.Variable region of light chain is shown among Figure 16 A, and variable region of heavy chain is shown among Figure 16 B.

The specific embodiment

1. general introduction

The invention provides the method for treatment, wherein treating according to priority interim or side by side giving the anti-CEA antibody of naked III class or its fragment and at least a therapeutic agent.This method is particularly useful for the treatment medullary thyroid carcinoma, but for treatment non-medullary thyroid carcinoma, colorectal carcinoma, hepatocarcinoma, cancer of pancreas, breast carcinoma, pulmonary carcinoma, head and neck cancer, bladder cancer, uterus carcinoma and ovarian cancer and even the cancer of high level expression CEA is not useful astoundingly yet.For example, consider in cancer, to treat with the horizontal expression CEA of 100ng/ gram tissue at least.This method further provides the compositions that comprises anti-CEA antibody of III class or antibody fragment, and wherein antibody does not have coupling each other with therapeutic agent or is connected.As used herein, phrase " the anti-CEA of III class " antibody or antibody fragment mean not react in conjunction with the antibody of CEA antigen (or CD66e) or fragment and with normal cross reacting antigen (NCA), meconium antigen (MA), granulocyte and CD66a-d (to be seen, Primus etc., U.S. Patent number 4,818,709, be incorporated herein by reference).The anti-CEA antibody of naked III class or its fragment can be humanized, chimeric, people or murine antibody.In a preferred embodiment, the anti-CEA antibody of naked III class or its fragment are humanized MN-14 antibody or its fragment.

Surprisingly, compositions as herein described and method also for the non-medullary thyroid carcinoma of treatment, comprise that colorectal carcinoma, cancer of pancreas, breast carcinoma, pulmonary carcinoma, hepatocarcinoma, bladder cancer, head and neck cancer and ovarian cancer are useful.Because the cancer of this class form is expressed less CEA than medullary thyroid carcinoma, thus beat all be that the combination meeting of anti-CEA antibody of naked III class and therapeutic agent is useful to treating non-medullary thyroid carcinoma.

The mechanism of the anti-CEA antibody of naked III class kill tumor cell is not known definitely and may comprises several mechanism.Suppose independent naked antibody or with therapeutic agent combination can by blocking-up its separately antigenic biologic activity or by stimulating natural immunity function, influence growth of tumor as the cell-mediated cytotoxicity (ADCC) of antibody dependent or the lysis of complement-mediated.In addition, independent naked antibody or combine and transfer activity to take place, to suppress and/or influence tumor cell adhesion to treat and control cancer by cell growth inhibiting and cell cycle progression, cell death inducing, inhibition blood vessel with therapeutic agent.In fact, anti-CEA antibody of the present invention or its fragment can be more more effective than treatment primary carcinoma disease in the treatment metastasis, because metastasis may be more responsive for the antagonist of tumor cell adhesion.This Therapeutic Method provides a kind of treatment plan, and it can provide effective therapeutic scheme to be optimized so that the anti-tumor activity to the individual patient maximum to be provided by allowing the different therapeutic agents with one or more of antagonist to carry out titration.

In one aspect of the invention, the anti-CEA antibody of naked III class or its fragment and therapeutic agent can replenish at least a extra therapeutic agent, as naked or link coupled antibody humanized, Mus, chimeric or the people, fusion rotein or its fragment.For example, another kind of non-barrier and not in conjunction with III class CEA antibody or the antibody fragment of granulocyte or CD66a-d; Non-barrier and not in conjunction with anti-CEA antibody of the II class of granulocyte or CD66a-d or antibody fragment; Or, can be used as therapeutic agent and carry out therapeutic alliance with preferred humanization MN-14 antibody at epi-position relevant or antigenic antibody with various cancers.Introduce more in detail as following, this extra antibody, fusion rotein or its fragment can be in conjunction with CEA or the another kind of antigens relevant with cancer or tumor.

2. definition

In ensuing introduction, used many terms and provide following definition to help to understand the present invention.

As described herein, Antibody isRefer to a kind of total length (promptly, naturally occurring or formed by normal immunoglobulin gene fragment regrouping process) immunoglobulin molecules is (for example, a kind of IgG antibody) or a kind of immunocompetence of immunoglobulin molecules (that is, specificity in conjunction with) part, such as a kind of antibody fragment.

" Antibody fragment" be the part of antibody, as F (ab ') ₂, F (ab) ₂, Fab ', Fv, scFv (strand Fv) etc.Do not consider structure, antibody fragment combines with the same antigen of being discerned by complete antibody.

Term " antibody fragment " also comprises the albumen of any synthetic or genetically engineered mistake, and it is by playing a role as antibody in conjunction with forming complex with specific antigen.For example, antibody fragment comprises the isolated fragment of being made up of the variable region, as " Fv " fragment of forming by heavy chain and variable region of light chain, the single chain polypeptide molecule (" sFv albumen ") of the wherein reorganization that is connected by peptide bond of light chain and variable region of heavy chain and the atom of forming by the amino acid residue of simulation hypervariable region.The Fv fragment can make up by different way with produce multivalent and/or the bonded form of polyspecific.For the former multivalent, they with react at CEA epi-position and more than one binding site, and for the polyspecific form, in conjunction with more than one epi-position (both can be the epi-position of CEA, also can be at CEA with a kind of different antigenic).

As used herein, term Antibody componentComprise complete antibody, fusion rotein and fragment thereof.

Naked antibodyNormally a kind of not with the link coupled complete antibody of therapeutic agent.Why so be because the Fc of antibody molecule partly provides effector or immunologic function, as complement fixation and ADCC (cytotoxicity that antibody relies on), it makes and can cause the mechanism of lysis to be implemented.Yet, the Fv part may not be that the treatment function of antibody is necessary, but other mechanism, as apoptosis, angiogenesis inhibitor, antimetastatic activity, anti-adhesion activity as to abnormal shape or the adherent inhibition of homotype with can be played a role and disturb the process of disease to the interference of signal pipeline.Naked antibody comprises polyclone and monoclonal antibody and fragment thereof, comprises murine antibody and some recombinant antibodies, as chimeric, humanized or people's antibody and fragment thereof.As defined in the present invention, " naked " and " not link coupled " synonym, and mean not with it that the therapeutic agent of administration is connected or coupling.

Chimeric antibodyBe a kind of albumen of reorganization, it contains the variable region of heavy chain of antibody and light chain, comprises deriving from species, is preferably the complementary determining region (CDRs) and the variable domains of rodentine antibody, and the constant region of antibody molecule then derives from those of people's antibody.Use for the veterinary, the constant region of chimeric antibody can derive from other species, as cat or Canis familiaris L..

Humanized antibodyBe a kind of recombiant protein, wherein derive from the antibody of species, the CDRs of the antibody of Rodents is for example transferred in people's heavy chain and the variable region of light chain from the heavy chain of Rodents antibody and light chain.The constant region of antibody molecule derives from those of people's antibody.

People's antibodyBe a kind of by the antibody that transgenic mice obtained, it has carried out " through engineering approaches " reacts on the antigenicity invasion and attack with generation human antibodies specific.In present technique, people's heavy chain and the importing of light chain gene seat element are derived from the mouse species of embryonic stem cell line, described embryonic stem cell line comprises the endogenous heavy chain of targeting and the destruction in light chain site.Transgenic mice can synthesize the people antibody special to the human antigen, and this mice can be used to produce the hybridoma of secretion people antibody.Green etc., Nature Genet.7:13 (1994), Lonberg etc., Nature 368:856 (1994) and Taylor etc., Int.Immun.6:579 (1994) have introduced the method that obtains people's antibody from transgenic mice.Can also make up people's antibody completely by gene or chromosome transfection method and display technique of bacteriophage, all these technology all are known in the art.For example see, McCafferty etc., Nature 348:552-553 (1990) is about from from gene repertoire produced in vitro people's antibody or its segmental description of the immune globulin variable region of epidemic disease donor rather.In present technique, will be cloned into the big or little coat protein gene of filobactivirus in the antibody variable gene frame, and on the phage particle surface, be shown as the functional antibodies fragment.Because the filobactivirus granule comprises the single stranded DNA copy of phage genome, also causes coding is showed the screening of the antibody gene of those characteristics based on the screening of this antibody function characteristic.In this way, some characteristics of phage simulation B cell.Phage display can carry out with many forms, about their summary, sees for example Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).

People's antibody can also be by external activated B cells produce.See U.S. Patent number 5,567,610 and 5,229,275, their full content is incorporated herein by reference.

Therapeutic agentBe a kind of individually, with antibody component be antibody or antibody fragment, or its subfragrnent is useful in treatment of diseases together simultaneously or the molecule or the atom of sequential administration.The example of therapeutic agent comprises antibody, antibody fragment, immune conjugate, medicine, cytotoxic agent, toxin, nuclease, hormone, immunomodulator, chelating agen, boron compound, photosensitizer or dyestuff, radiosiotope or radionuclide, immune conjugate or its combination.

Immune conjugateIt is a kind of and the link coupled antibody component of therapeutic agent.Suitable therapeutic agent as mentioned above.

As used herein, term Antibody fusion proteinBe a kind of antigen binding molecules of recombinant production, wherein two or more identical or different natural antibodys, single-chain antibody or have identical or different specific antibody fragment and partly link to each other.The anti-CEA fusion rotein of III class comprises at least one CEA binding site.Preferably, the anti-CEA fusion rotein of III class is the MN-14 fusion rotein.

The valence state of fusion rotein shows that the brachium conjunctivum of fusion rotein or site have antigen or epi-position; Be unit price, bivalence, trivalent or polyvalent.The polyvalency of antibody fusion protein mean can utilize with multiplephase mutual effect during antigen combines, thereby enhancing and antigen or the bonded affinity of synantigen not.Specificity shows that antibody fusion protein can be in conjunction with how many kinds of dissimilar antigen or epi-position; That is, monospecific, bispecific, tri-specific, polyspecific.Utilize these definition, natural antibody, for example IgG is a bivalence, because it has two brachium conjunctivums, but is monospecific, because it is in conjunction with one type antigen or epi-position.A kind of polyvalent fusion rotein of monospecific has more than one binding site for same antigen or epi-position.For example, the double antibody of monospecific is the fusion rotein with two binding sites that react with same antigen.Fusion rotein can comprise the multivalence or the polyspecific combination of a plurality of copies of different antibodies component or same antibody component.Fusion rotein can also comprise therapeutic agent.

ImmunomodulatorIt is the defined in the present invention therapeutic agent when presenting, change, suppressing or stimulating the health immune system.Usually, useful in the present invention immunomodulator immune stimulatory cell proliferation or be activated in the immunoreation cascade is as macrophage, B cell and/or T cell.An example of immunomodulator as described herein is a kind of cytokine, it be a kind of population of cells (for example, the T lymphocyte that excites) the solubility small protein matter of the about 5-20kDa that discharges after the contact specific antigen, it plays a role as the intercellular medium between the cell.Will appreciate that as those skilled in the art the example of cytokine comprises that lymphokine, monokine, interleukin and several relevant signals transmit molecule, as tumor necrosis factor (TNF) and interferon.Chemotactic factor is a subclass of cytokine.Some interleukin and interferon are the examples that stimulates the cytokine of T cell or other immune cell propagation.

Monoclonal antibody comprises the preparation of chimeric, humanized and people's antibody

Monoclonal antibody (MAbs) is that the community of interest of antibody of specific antigen and this antibody only comprise one type antigen binding site and an epi-position on the conjugated antigen determinant only.Can obtain the Rodents monoclonal antibody of specific antigen by method known in those skilled in the art.See, for example, Kohler and Milstein, (editors) such as Nature256:495 (1975) and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, VOL.1,2.5.1-2.6.7 page or leaf (John Wiley and Sons 1991) [hereinafter " Coligan "].In brief, monoclonal antibody can by with contain antigenic compositions injection mice, check the existence of antibody product by removing blood serum sample, remove spleen with obtain bone-marrow-derived lymphocyte, bone-marrow-derived lymphocyte is merged mutually with the myeloma cell produce hybridoma, clone this hybridoma, select to produce positive colony at this antigenic antibody, cultivate generation at the clone of this antigenic antibody and from the hybridoma culture separation antibody obtain.

Technology by many abundant foundation can be separated from the hybridoma culture and purification MAb s.This isolation technics comprises affinity chromatography, size exclusion chromatography method and the ion exchange chromatography of use albumen-A agarose gel.Referring to, for example, Coligan is in 2.7.1-2.7.12 page or leaf and 2.9.1-2.9.3 page or leaf.Also referring to Baines etc., " purification of immunoglobulin G (IgG) ", molecular biological method, the 10th volume, the 79-104 page or leaf (The Humana Press, Inc.1992).

Produce antibody by known antibody production method at peptide backbone.For example, the immunogen in the injection complete Freund's adjuvant is as (peptide) _n-KLH, wherein KLH is a keyhole limpet hemocyanin, n=1-30, twice identical immunogen that is suspended in the incomplete Freund's adjuvant of injection in immune competence animal subsequently.Give the animal intravenous injection at last and strengthen antigen, then after three days, collect splenocyte.Subsequently the splenocyte of collecting is merged mutually with Sp2/0-Ag14 myeloma cell and utilize and directly the anti-reactive polypeptide of the clone's that obtains culture supernatants is analyzed in conjunction with ELISA.Utilize initial immunogenic fragments of peptides can analyze the good specificity that produces antibody.Utilize the automatic peptide synthesizer can prepare these fragments easily.For antibody producing, the hybridoma that separates enzyme defect is can screen the cell line of fusion.This technology can also be used for comprising at one or more chelate of linker, and for example In (III)-DTPA chelate is cultivated antibody.Monoclonal mouse antibody at In (III)-DTPA is known (authorizing the U.S. Patent number 5,256,395 of Barbet).

The another kind of method of producing antibody is to produce by the Ruzhong at breeding transgenic livestock.See, for example, Colman, A., Biochem.Soc.Symp., 63:141-147,1998; United States Patent (USP) 5,827,690 is incorporated herein by reference at this full content with the two.Prepare two kinds of constructs that contain the dna fragmentation of paired heavy chain immunoglobulin of coding and light chain respectively.Dna fragmentation is cloned in the expression vector that contains a promoter sequence, and described promoter sequence is preferably expressed in the mammal epithelial cell.Example includes but not limited to the promoter from rabbit, cattle and sheep casein gene, cattle α-lactoglobulin gene, sheep gene and mice whey acid protein gene.Preferably, the fragment of insertion is at the flank of holding from the homologous genes group sequence 3 ' of mammal specific gene.This provides polyadenylation site and has transcribed critical sequences.Expression cassette is injected the pronucleus of the mammalian ovum of fertilization altogether, subsequently this ovum transfer is gone in the uterus of the female body of receptor and allow it breed.After the birth, with regard to two kinds of genetically modified existence the offspring is screened by the Southern analysis.Antibody will exist, and heavy chain all must be expressed in identical cell with light chain gene simultaneously.Utilize standard immunoassay method known in the art to analyze with regard to the existence of antibody or antibody fragment and functional milk to the transgenic jenny.Can utilize standard method known in the art antibody purification from milk.

After beginning to cultivate, the variable gene of monoclonal antibody can be cloned from hybridoma, checked order and is prepared by recombinant technique subsequently at immunogenic antibody.For example, publication Orlandi etc., Proc.Nat ' l Acad.Sci.USA 86:3833 (1989) have introduced the conventional method of clone's mouse immune globulin variable zone, and the full content of the document is incorporated herein by reference.The humanization of murine antibody and antibody fragment and chimericization are known in those skilled in the art.Chimeric antibody is a kind of recombiant protein, and it comprises the variable region that comprises CDRs, and described CDRs is derived from the species of animal, as a kind of Rodents antibody, and the other parts of antibody molecule, promptly constant region then is derived from people's antibody.The use that is derived from the antibody component of humanization and chimeric monoclonal antibody has reduced the potential problem that is associated with Mus constant region immunogenicity.The technology that makes up chimeric antibody is known in those skilled in the art.As an example, Leung etc., Hybridoma 13:469 (1994), having described them is V by the LL2 monoclonal antibody of will encoding how _kAnd V _HThe DNA sequence of domain makes up with people k and IgG1 constant region domain respectively that to produce LL2 chimeric, and described LL2 monoclonal antibody is a kind of anti-CD22 antibody.

Sequence that can also be by the Mus FR in the variable region of replacing chimeric MAb with one or more different people FR is with chimeric monoclonal antibody (MAb) humanization.Particularly, by the mice complementary determining region is transferred in the people variable region by the heavy chain and the variable region of light chain of mouse immuning ball protein, and the people's residue that replaces subsequently in the framework region of Mus counterpart is produced humanized monoclonal antibody.Often cause weakening or even lose of antibody affinity among the people FRs owing to simply mice CDRs is transferred to, therefore may need extra modification so that the original affinity of recovery murine antibody.This can realize by the one or more people's residues in the FR district are replaced with the homologue of their Mus, so that obtain a kind of antibody that its epi-position is had the good binding affinity.See, for example, Tempest etc., Biotechnology 9:266 (1991) and Verhoeyen etc., Sciennce 239:1534 (1988).

In a preferred embodiment, some residues in humanized anti-CEA antibody or its fragment are replaced by their Mus homologue.In addition, known chimeric anti-CEA shows similar binding affinity to the homologue of its Mus, defective design in the initial form of the anti-CEA MAb of humanization, if any, can identify by those of the light chain of chimeric anti-CEA and heavy chain and humanization form are mixed mutually and mate.Preferably, humanized anti-CEA antibody is humanized MN-14 antibody, and its preparation and sequence be at U.S.5, introduces in 874,540, and its full content is incorporated herein by reference.Although two kinds of people's antibody REI and NEWM are used to prepare the preferred antibody of humanized and chimeric MN-14, can also with from the combination of the frame sequence of 2 kinds or multiple different people antibody as V _HAnd V _KJones etc., Nature321:522 (1986), Riechmann etc., Nature 332:323 (1988), Verhoeyen etc., Science239:1534 (1988), Carter etc., Proc.Nat ' lAcad.Sci.USA 89:4285 (1992), Sandhu, Crit.Rev.Biotech.12:437 (1992) and Singer etc., J Immun.150:2844 (1993) has introduced the production of humanization MAbs, hereby each piece document all is incorporated herein by reference.In addition, can be improved by the mutation of CDRs at humanized, chimeric and affinity people MAbs of a concrete epi-position, thus the same effective before the sudden change than the antibody of low dosage and higher dosage than the MAb of low-affinity.Referring to for example, W00029584A1.

In another embodiment, the anti-CEA monoclonal antibody of antibody behaviour III class of the present invention.Can from transgenic nonhuman animal, obtain anti-CEA MAb or another kind of people's antibody.See, for example, Mendez etc., Nature Genetics, 15:146-156 (1997) and U.S. Patent number 5,633,425 are incorporated herein by reference their full content.For example, can from transgenic mice, reclaim people's antibody with human immunoglobulin gene's seat.Preferably, anti-CEA antibody is MN-14 antibody.Come the mouse humoral immune system is carried out humanization by endogenous immunoglobulin gene of deactivation and importing human immunoglobulin gene seat.Human immunoglobulin gene's seat is very complicated and comprise a large amount of discontinuous fragments, and it has accounted for about 0.2% of people's gene group altogether.Can produce enough antibody constituent in order to ensure transgenic mice, the big fragment of people's heavy chain and light chain gene seat must be imported in the mice genome.This is achieved by a process progressively, and beginning to be formed in kind is the yeast artificial chromosome (YACs) of containing people's heavy chain or light chain immunoglobulin loci in the configuration.Because each inserts segmental size is about 1Mb, so YAC makes up the homologous recombination that needs the immunoglobulin loci overlapping fragments.The yeast spheroblast and the mouse embryo stem cell that contain YAC by fusion import the fusion mice respectively with two kinds of YAC, a kind of heavy chain gene seat that contains among described two kinds of YAC, and another kind contains the light chain gene seat.Subsequently clone's microinjection of embryonic stem cell is gone in the mice blastocyst.To chimeric malely screening of obtaining with regard to its ability by their embryonal system transhipment YAC, and with mice one hybridization of murine antibody production defective.Hybridize two kinds of transgenic strains, a kind of people's of containing heavy chain gene seat and another kind contains people's light chain gene seat has produced the offspring who reacts on immunity and produce people's antibody.

Can also import in the mouse embryo stem cell by the human immunoglobulin gene that the chromosome transfer (MMTC) of microcell mediation will not reset.See, for example, Tomizuka etc., Nature Genetics, 16:133 (1997).The microcell that contains human chromosome in the method merges mutually with mouse embryo stem cell.The chromosome that shifts keeps with being stabilized, and adult chimera shows suitable tissue specific expression.

As a kind of selection, antibody of the present invention or antibody fragment can come from the people antibody fragment of separation from the immunoglobulin combinatorial library.See, for example, Barbas etc., METHODS:ACompanion to Methods in Enzymology 2:119 (1991) and Winter etc., Ann.Rev.Immunol.12:433 (1994) is incorporated herein by reference them.Utilize phage display to pass through through engineering approaches and in escherichia coli (E.Coli), express antibody fragment and can overcome and the many difficulties that are associated by B cell immortalization generation monoclonal antibody.In order to ensure the recovery of high-affinity monoclonal antibody, the immunoglobulin combinatorial library must comprise big whole constituents.A kind of typical strategy utilizes the mRNA by lymphocyte of the mice of immunity or splenocyte acquisition to utilize reverse transcription to synthesize cDNA.By PCR increase respectively heavy chain and light chain gene and connect into the phage clone carrier.Produce two kinds of different libraries, a kind of heavy chain gene that contains, one and contain light chain gene.From every kind of library, separate phage DNA, heavy chain and sequence of light chain are linked together and pack to form combinatorial library.Each phage all comprises the random pair of heavy chain and light chain cdna s, and instructs the expression of antibody chain in the infection cell after colibacillary infection.In order to identify the antigenic antibody of recognition objective, phage library is coated with flat board, the antibody molecule that will be present in the plaque is transferred to filter.With filter with radiolabeled antigen incubation and wash subsequently to remove unnecessary unconjugated part.Radioactivity speckle on radioautogram has been identified the speckle that contains in conjunction with this antigenic antibody.Can be for example, (La Jolla, CA) middle acquisition is for producing useful clone and the expression vector of human normal immunoglobulin's phage library from STRATAGENE Cloning Systems.

In one embodiment, as in Hansen etc., U.S. Patent number 5,874,540; Hansen etc., Cancer, 71:3478 (1993); Primus etc., U.S. Patent number 4,818,709 and Shively etc., U.S. Patent number 5,081, that is introduced in 235 produces antibody of the present invention, and the full content of these documents is incorporated herein by reference.

The production of antibody fragment

The present invention considers the anti-CEA antibody of III class, the segmental application of preferred MN-14 antibody.The anti-CEA antibody of III class of the present invention or its fragment be not in conjunction with granulocyte or 66a-d.The antibody fragment of identification specificity epi-position can be produced by known technology.For example, can prepare antibody fragment by the Proteolytic enzyme of antibody or by this segmental DNA expression in escherichia coli of encoding.Antibody fragment is a kind of antigen-binding portion thereof of antibody, as F (ab ') ₂, Fab ', Fab, Fv, scFv etc., can carry out pepsin to complete antibody and papain digestion obtains by traditional method.

For example, can be called F (ab ') to provide by carry out enzyme action with pepsin ₂The fragment of 100Kd produce antibody fragment.This fragment can be utilized a kind of thiol reductant, and randomly a kind of blocking groups of sulfydryl further cuts, to produce the Fab ' unit price fragment of 50Kd.Perhaps, utilize the enzyme action of papain directly to produce two unit price Fab fragments and a Fc fragment.Goldenberg, U.S. Patent number 4,036,945 and 4,331,647 and the list of references that comprises wherein these methods are introduced, the full content with these patents is incorporated herein by reference here.Also see Nisonoff etc., ArchBiochem.Biophys.89:230 (1960); Porter, Biochem.J 73:119 (1959), Edelman etc., in METHODSIN ENZYMOLOGY VOL.1,422 pages (academic press 1967) and Coligan are in 2.8.1-2.8.10 and 2.10.-2.10.4 page or leaf.

Can also use the method for other cutting antibody, as the separation of heavy chain with form unit price light-heavy chain fragment, further cutting fragment maybe can use other zymetology, chemistry or genetic technology, as long as fragment can be in conjunction with the antigen of being discerned by complete antibody.

For example, the Fv fragment comprises V _HAnd V _LThe association body of chain.As at Inbar etc., described in Proc.Nat ' l.Acad.Sci.U.S.A.69:2659 (1972), this association can be non-covalent.Perhaps, variable chains can or be undertaken crosslinked by chemical substance such as glutaraldehyde by the intermolecular disulfide bond connection.See, for example, Sandhu, Crit.Rev.Biotech.12:437 (1992).

Preferably, the Fv fragment comprises the V that connects by peptide bond _HAnd V _LChain.By making up a kind of coding V that connects by oligonucleotide that contains _HAnd V _LThe structural gene of the DNA sequence in district prepares these single chain antigen binding protein matter (sFv).This structural gene is inserted in a kind of expression vector, subsequently described expression vector is imported a kind of host cell, in escherichia coli.The synthetic a kind of single chain polypeptide of the host cell of reorganization has a connection peptides between two V districts.Whitlow etc., Methods:A Companion to Methods in Enzymology, 2:97 (1991) have introduced the method for producing sFvs.Also see Bird etc., Science 242:423 (1988), Ladner etc., U.S. Patent number 4,946,778; Pack etc., Bio Technology 11:1271 (1993) and Sandhu, the same.

The another kind of form of antibody fragment is the peptide of the single complementary determining region of coding (CDR).CDR is a section of antibody variable region, and structurally the remainder in complementation and variable ratio district is variable more mutually with the epi-position of antibodies for it.Therefore, CDR some the time be called the hypervariable region.A variable region comprises three CDRs.The CDR peptide can be by making up the coding target antibody the gene of CDR obtain.This gene for example is by utilizing the polymerase chain reaction to be prepared with the variable region of synthetic RNA from antibody producing cells.Referring to for example, Larrick etc., Methods:A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, " Genetic Manipulation of Monoclonal Antibodies; " in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICALAPPLICATION, Ritter etc. (editor), 166-179 page or leaf (Cambridge University Press 1995); With Ward etc., " Genetic Manipulation and Expression of Antibodies, " in MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, Birch etc. (editor), the 137-185 page or leaf (Wiley-Liss, Inc.1995).

Can also use the method for other cutting antibody, as separate heavy chain with form unit price light-heavy chain fragment, further cutting fragment, or other zymetology, chemistry or genetic technology is as long as fragment can be in conjunction with the antigen of being discerned by complete antibody.

The humanization of treatment usefulness, chimeric and the anti-CEA antibody of people

What introduced among the present invention is compositions and the method for utilizing Mus, chimeric, the humanized and anti-CEA antibody of people III class or its fragment to treat.Preferably, the anti-CEA antibody of III class or its fragment are MN-14 antibody or its fragment.Antibody of the present invention can be used for the treatment of medullary thyroid carcinoma (MTC), and the cancer of the expression CEA of non-MTC.The cancer of the expression CEA of exemplary non-MTC comprises colorectal carcinoma, cancer of pancreas, hepatocarcinoma, gastric cancer, pulmonary carcinoma, head and neck cancer, bladder cancer, uterus carcinoma, breast carcinoma and ovarian cancer.

Compositions

What this paper considered is the compositions that contains the anti-CEA monoclonal antibody of at least a III class (MAb) or its fragment and at least a therapeutic agent, described antibody and therapeutic agent not coupling mutually each other, and thereby be present in the compositions as the non-coupling form of every kind of component.In the antibody that comprises more than one or its fragment, in the compositions as second kind of anti-CEA antibody of III class, this second kind of antibody is (that is, can not block the combination of first kind of anti-CEA antibody of III class or antibody fragment) of non-barrier.

In one embodiment, the anti-CEA monoclonal antibody of III class or its fragment are humanized, chimeric or people completely, wherein humanized, chimeric, or people MAb keeps the anti-CEA binding specificity of III class of the anti-CEA MAb of Mus III class basically completely.

In a preferred embodiment, the anti-CEA monoclonal antibody of III class or its fragment are MN-14 antibody or its fragment.Preferably, MN-14 monoclonal antibody or its fragment comprise the complementary determining region (CDRs) of Mus MN-14 monoclonal antibody, and the CDRs of the variable region of light chain of wherein said MN-14 monoclonal antibody comprises the CDR1 that contains aminoacid sequence KASQDVGTSVA; The CDR2 that contains aminoacid sequence WTSTRHT; With the CDR3 that contains aminoacid sequence QQYSLYRS; And the CDRs of the variable region of heavy chain of the anti-CEA antibody of described III class comprises the CDR1 that contains TYWMS; The CDR2 that contains EIHPDSSTINYAPSLKD; With the CDR3 that contains LYFGFPWFAY.Also will be preferably, MN-14 monoclonal antibody and CEA react and do not react with normal cross reacting antigen (NCA) and meconium antigen (MA).But, can be used for the therapeutic alliance carried out together with the CEA specific antibody at the antibody of these cross reactivity determinants, as making up with the MN-14 monoclonal antibody.

In another embodiment of the invention, MN-14 monoclonal antibody or its fragment are humanized or people MN-14 antibody or its fragment completely.The framework region (FRs) of humanization MN-14 antibody or its segmental light chain and variable region of heavy chain preferably comprises the aminoacid of at least one replacement from the corresponding FRs of Mus MN-14 monoclonal antibody.Also will be preferably, humanization MN-14 antibody or its fragment comprise the aminoacid of the corresponding FR of Mus MN-14 antibody, are selected from the amino acid residue 24,28,30,48,49,74 and 94 of the Mus variable region of heavy chain (KLHuVhAIGA) of Figure 14 A-C as mentioned above or Figure 15 B or Figure 16 B.The aminoacid sequence of preferred humanization variable region of heavy chain is also at Hansen etc., and U.S. Patent number 5,874 provides in 540, and its full content is incorporated herein by reference.Also will be preferably, the humanization variable region of heavy chain comprises the aminoacid sequence shown in Figure 14 A-C, is expressed as KLHuVhAIG and KLHuVhAIGAY.In another embodiment, humanization MN-14 antibody or its fragment comprise at least one aminoacid from the corresponding FR of Mus MN-14 variable region of light chain.The most preferably, humanized MN-14 antibody or its fragment comprise the variable region of light chain of Figure 13 A or Figure 15 A or Figure 16 A.Another embodiment of the invention is a kind of compositions that comprises chimeric MN-14 monoclonal antibody or its fragment and at least a therapeutic agent, described antibody and therapeutic agent not coupling mutually each other, and thereby be present in the compositions as the non-coupling form of every kind of component.Preferably, chimeric MN-14 antibody or its fragment comprise the CDRs of the Mus MN-14 variable region of light chain shown in Figure 13 A or Figure 15 A or Figure 16 A and the CDRs of the Mus MN-14 variable region of heavy chain as shown in Figure 14 A-C or Figure 15 B or Figure 16 B.

This paper has also introduced a kind of naked Mus, humanization, the anti-CEA antibody of III the class chimeric or people or its fragment and a kind of therapeutic agent of comprising, and second kind of compositions naked or the anti-CEA antibody of link coupled III class or its antibody fragment, described second kind of antibody or its antibody fragment are non-barrier, promptly, can not block first kind of anti-CEA antibody of III class or its segmental combination, and in a kind of pharmaceutically useful carrier, prepare.In other words, the anti-CEA antibody of III class or its fragment be for all being non-barrier each other, thereby allow antibody or its fragment in conjunction with CEA (66e).In addition, III class CEA antibody of the present invention or its antibody fragment, and be used for those of therapeutic alliance, not in conjunction with granulocyte or CD66a-d.Be included in Kuroki etc. with the anti-CEA antibody of naked III class of antibody fragment of the present invention as naked antibody or as other III antibody-like that the component of immune conjugate is suitable for therapeutic alliance, JP R Cancer Res., 78 (4): 386 (1987) and Hammarstrom (Cancer Res.52 (8): the non-blocking antibody 2329 (1992)) or its fragment, it is not also in conjunction with granulocyte or CD66a-d.

In addition, other anti-CEA antibody as the II antibody-like, can be used for and the anti-CEA antibody combination of III class of the present invention with naked or link coupled form.This can as the II antibody-like of therapeutic alliance or antibody fragment be non-barrier and not in conjunction with granulocyte or CD66a-d.For example, one or more chimeric or the anti-CEA antibody of humanized II class or its fragments, as MN-6 or NP-3, can with the anti-CEA antibody of a kind of III class of the present invention or its fragment combination.These two kinds of antibody do not react (Hansen etc., Cancer 1993Jun 1 with CD66a-d or granulocyte; 71 (11): 3478-85).Many publications disclose the MAbs of identification CEA and CEA gene family different members, as Thomps on etc., and J Clin.Lab.Anal.5:344 (1991); Kuroki etc., J BioL Chem.266:11810 (1991); Nagel etc., Eur.J Biochem.214:27 (1993); Skubitz etc., J Immunol.155:5382 (1995); Skubitz etc., J.Leukoc.Biol.60:106 (1996); With Chen etc., Proc.Natl.Acad.Sci.93:14851 (1996).

And second antibody or antibody fragment are not coupling (naked) or link coupled with at least a therapeutic agent (immune conjugate).Can prepare immune conjugate by indirectly a kind of therapeutic agent being coupled to a kind of antibody component.At Shih etc., Int.R Cancer, 41:832 (1988); Shih etc., Int.J Cancer, 46:1101 (1990); With Shih etc., U.S. Patent number 5,057 has been introduced general technology in 313.General method relates to antibody component and the reaction of a kind of carrier polymer that has the sugar moieties of oxidation with a kind of, and described carrier polymer has at least a free amine functions and loaded multiple medicine, toxin, chelating agen, boron additament or other therapeutic agent.This reaction causes initial Schiff alkali (imines) key, and it can carry out stabilisation to form final conjugate by being reduced into secondary amine.Preferably, the anti-CEA antibody in the compositions that is used for the treatment of or its fragment are MN-14 antibody or its fragment.More preferably, MN-14 antibody or its fragment are humanized.

The present invention also considers a kind of the contain naked anti-CEA antibody of III class humanized, chimeric, Mus or the people or its fragment and a kind of therapeutic agent, and a kind of for example link coupled or non-link coupled antibody or its segmental second therapeutic agent, described antibody is not the anti-CEA antibody of II class or III class.In one embodiment, second therapeutic agent (or second kind of antibody or its fragment) right and wrong link coupled (naked) or be coupled at least a therapeutic agent.Be suitable for the non-I I class of therapeutic alliance or the anti-CEA antibody of III class and fragment thereof and include, but not limited to cancer associated antibodies and fragment thereof.The example of cancer associated antibodies and antibody fragment is in conjunction with EGP-1, EGP-2 (as 17-1A), MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn, Thomson-Friedenreich antigen, neoplasm necrosis antigen, VEGF or other tumor vessel generation antigen, Ga 733 or its combination.As discussed above, can not be used in combination with III class CEA antibody in conjunction with the II class and the anti-CEA MAbs of III class of CD66a-d or granulocytic non-barrier yet.Be suitable for other antibody of therapeutic alliance and antibody fragment also comprises targeting oncogene labelling or product those, or at the tumor vascular system labelling, as the antibody of angiogenesis factor, placental growth factor (P1GF) with at the antibody of some immune response modifier, as antibody at CD40.

Method

Also introduced the method for treatment medullary thyroid carcinoma and non-medullary thyroid carcinoma among the present invention.Non-medullary thyroid carcinoma comprises the tumor of colorectal carcinoma and any other expression CEA, as cancer of pancreas, breast carcinoma, hepatocarcinoma, ovarian cancer, and pulmonary carcinoma, head and neck cancer, carcinoma of endometrium, bladder cancer and the hepatocarcinoma of the CEA of the expression variable number of some type.The level of CEA is than being present in much lower in the medullary thyroid carcinoma in the cancer of these types, thereby but necessary be that the anti-CEA therapy of the enough height III of level class of CEA provides effective treatment.But normal mucous membrane of colon layer has about 500ng/ gram is suitable for treating with method of the present invention with the cancer of the horizontal expression CEA of 100ng/ gram tissue.

For example, consider a kind of method for the treatment of medullary thyroid carcinoma or non-medullary thyroid carcinoma herein, comprise simultaneously or treat the anti-CEA monoclonal antibody of III class or its fragment and at least a therapeutic agent of effective dose according to priority, and choose wantonly in a kind of pharmaceutically useful carrier and prepare to the experimenter.Preferably, the anti-CEA monoclonal antibody of III class or its fragment are chimeric, Mus, humanized or the people, and wherein chimeric, the anti-CEA MAb of III class humanized, Mus or the people has kept the binding specificity of the anti-CEA antibody of III class of Mus MAb basically.More preferably, the anti-CEA antibody of III class is humanized, most preferably, is as this paper and United States Patent (USP) 5,874, the humanized MN-14 monoclonal antibody described in 540.Preferably a kind of cytotoxic agent of therapeutic agent, being more preferably is a kind of alkylating agent, most preferably is dacarbazine (DTIC).The antineoplastic cytostatics and the cytotoxic agent of other classification also can be used for and these antibody combinations as CPT-11, particularly in the treatment of colorectal carcinoma.But in another embodiment, therapeutic agent can not be DTIC also.

This paper also considers a kind of method for the treatment of medullary thyroid carcinoma or non-medullary thyroid carcinoma, comprise simultaneously or treat the anti-CEA monoclonal antibody of first kind of III class or its fragment and at least a therapeutic agent of effective dose in order to the experimenter, and naked or link coupled second kind of humanized, chimeric, the people's or Mus monoclonal antibody or its fragment, and choose wantonly in a kind of pharmaceutically useful carrier and prepare.Preferably, first kind of anti-CEA MAb of III class is humanized MN-14 antibody or its fragment.In one embodiment, second kind of antibody or its fragment (i.e. second therapeutic agent) are relevant antibody or its fragments of cancer, are selected from and TAG-72, EGFR, HER2/neu, MUC1, MUC2, MUC3, MUC4, EGP-1, EGP-2, AFP, Tn or another kind of aforesaid this class antigen reactive monoclonal antibody or its fragment relevant with tumor.In another embodiment, second kind of antibody or its fragment can be the anti-CEA antibody of a kind of different IIII class or its fragment, it be non-barrier and not in conjunction with granulocyte or CD66a-d.

In another embodiment, second kind of anti-CEA antibody is a kind of II antibody-like or its fragment, and as at described in Hammarstrom and the Kuroki those, condition is that they do not combine with granulocyte or CD66a-d.Antibody and fragment thereof can be simultaneously or administration or with the therapeutic agent administration according to priority.In one embodiment, second kind of antibody or its fragment can be naked also can being coupled on a kind of therapeutic agent.

Therefore, the present invention considers according to priority or side by side gives Mus, humanized, chimeric and anti-CEA antibody and fragment thereof the people with one or more therapeutic agents, or carries out administration as the multi-mode therapy.Multi-mode therapy of the present invention comprises the immunization therapy of using the anti-CEA antibody of a kind of III class or its fragment and a kind of therapeutic agent, replenishes with a kind of non-link coupled or link coupled antibody, non-link coupled or link coupled fusion rotein or its segmental administration.For example, a kind of non-link coupled humanized, chimeric, MN-14MAb Mus or the people or its fragment can with naked humanized of another kind, Mus, the anti-CEA antibody of III the class chimeric or people (as a kind of at CEA go up different epi-positions and also not in conjunction with the antibody of granulocyte or CD66a-d) combination, or with a kind of humanized, chimeric, the immune conjugate combination of the anti-CEA antibody of III class Mus or the people, described immune conjugate is coupled to a kind of radiosiotope, chemotherapeutics, cytokine, enzyme, enzyme inhibitor, hormone or hormone antagonist, metal, toxin or its combination.The anti-CEA antibody of naked III class or its fragment also can make up with a kind of link coupled or non-link coupled Mus fusion rotein, the anti-CEA antibody of III class humanized, chimeric or the people.But, the anti-CEA antibody of III class that is used for therapeutic alliance be each other non-barrier and can not be in conjunction with granulocyte or CD66a-d.Preferably, with the anti-CEA antibody of naked III class and second kind naked or link coupled antibody, fusion rotein or its fragment carry out administration according to priority or simultaneously.Also will be preferably, being used for one of the antibody of therapeutic alliance or antibody fragment is a kind of naked humanized MN-14 antibody or its fragment.In addition, the second antibody of using as naked or link coupled antibody, fusion rotein or its fragment can be II class CEA antibody or its fragment of a kind of people, humanized, chimeric or Mus, it be non-barrier and can not be in conjunction with granulocyte or CD66a-d.

In method as herein described, the experimenter accepts the anti-CEA antibody of a kind of naked III class or its fragment at least, it be before a kind of therapeutic agent, afterwards or administration therewith.Preferably, therapeutic agent is the medicine that is used for the cancer chemotherapy of standard, as the oxaliplatin medicine in the taxane in the ovarian cancer or platinum medicine, the colorectal carcinoma, is used in the gemcitabine in cancer of pancreas and other cancer, or is used in the Taxane derivative in the breast carcinoma.Cox 2 inhibitor is still represented another kind of medicament, demonstration is active with typical cytotoxic agent is combined in cancer chemotherapy for it, and can using in the same way in the present invention, but the antibody that combination is correlated with other cancer with medicament except independent CEA antibody and combination.Randomly, these medicines can be united use with radiolabeled antibody, and the existing CEA antibody coupling matter of described radiolabeled antibody also has and the link coupled radioactivity conjugate of other associated antibodies of mentioned kind.Also preferably, the anti-CEA antibody of III class or its fragment are MN-14 antibody or its fragment.Also will be preferably, MN-14 antibody or its fragment are humanized.

In a preferred embodiment, the anti-CEA antibody of naked III class or its fragment and dacarbazine (DTIC), AC or vincristine, or these combination in any according to priority (before or after) or side by side carry out administration.For example, DTIC and cyclophosphamide can carry out administration according to priority or side by side with the anti-CEA antibody of naked III class or its fragment.Preferably, anti-CEA antibody or its fragment are humanized MN-14 antibody or its fragment.Similarly, 5-fluorouracil combination is a kind of therapy that is used for the treatment of colorectal carcinoma with the independent or combination folinic acid with Irinotecan (CPT-11).Other suitable combined chemotherapy therapy is known for those skilled in the art, and as the independent oxaliplatin of using, or combination is with these other medicine.Therefore, according to used therapy, can both be used for the treatment of MTC or non-MTC with any of these chemotherapeutics and the anti-CEA antibody of naked III class or its segmental conjoint therapy.In medullary thyroid carcinoma, preferred other chemotherapeutics still, as a kind of alkylating agent (for example, DTIC), and gemcitabine and other cytotoxic agent of classification more recently.Chemotherapeutics and the anti-CEA antibody of naked III class or its fragment can be with any order or administrations together.In other words, antibody and therapeutic agent can simultaneously or carry out administration according to priority.In a preferred multi-mode therapy, give a kind of according to link coupled or non-link coupled anti-CEA antibody of the present invention, fusion rotein or its fragment before, give chemotherapeutics and the anti-CEA antibody of naked III class or its fragment afterwards or simultaneously.Preferably, the anti-CEA antibody of III class or its fragment are humanization MN-14 antibody or its fragment.

A kind of therapeutic scheme of preferred multi-mode therapy is to give hMN-14 and DTIC 3 days, only gives hMN-14 at the 7th, 14,21 day, and is administered once in per subsequently 21 days, continues 12 months treatment phase.The dosage of hMN-14 is 0.5-15mg/kg body weight/each infusion, be more preferably 2-8, and still be more preferably 3-5mg/kg body weight/each infusion, and the preferred dose of the dosage of DTIC such as present clinical practice, but also 2/3 of the maximum preferred dose that can use or still less, reduce the side effect relevant thus with medicine.Can give multiple medicine circulation, as every 3-6 month, be accompanied by lasting naked Antybody therapy, or be accompanied by the antibody of radiolabeled antibody, drug coupling, and the different schemes that comprises some cytokine such as G-CSF and/or GM-CSF, thereby adjust every kind of dosage the toxicity for patient is not strengthened by therapeutic combination.The cytokine somatomedin, application as G-CSF, can make and to give even the myelosuppressive of high dose more, become possibility as radiolabeled antibody or cytotoxic drug, and these schemes and dosage will individually be adjusted patient according to patient's morbid state and treatment in the past, and all morbid states and treatment in the past all influence the bone marrow state and for the toleration of other cytotoxicity treatment.In a preferred embodiment, the dosage with 100-600 milligram protein/agent/injection gives MN-14 antibody or its fragment.Still will be preferably, give MN-14 antibody or its fragment, preferred multiple dosage with the dosage of 300 milligrams of protein/agent/injections.

Therapeutic agent

Here cited therapeutic agent is at those medicaments also useful with naked antibody separate administration as described herein.Suitable therapeutic agent can be selected from cytotoxic agent, radionuclide, immunomodulator, photosensitive therapeutic agent (as chromogen or dyestuff), immune conjugate, another kind of naked antibody, hormone, or its combination.Therapeutic agent comprises, for example chemotherapeutics such as vinca alkaloids and other alkaloid, anthracycline, epipodophyllotoxin, taxane, antimetabolite, alkylating agent, antibiotic, cox 2 inhibitor, antimitotic agent, anti-angiogenic agent and apoptosis agent, particularly amycin, methotrexate, paclitaxel, CPT-11, camptothecine and from other medicament of the anticarcinogen of these and other classification etc.Other comprises chlormethine, alkyl sulfonic ester, nitroso ureas, triazenes, folacin, cox 2 inhibitor, pyrimidine analogue, purine analogue, iridium-platinum complex, hormone, toxin (for example, ribonuclease, Pseudomonas exotoxin) etc. for preparation immune conjugate and the useful cancer chemotherapeutic drug of antibody fusion protein.According to the difference of the malignant tumor that will treat, preferred therapeutic agent comprises DTIC, CPT-11,5-fluorouracil, paclitaxel, oxaliplatin, AC and vincristine, or its combination.More preferably, amycin, DTIC, cyclophosphamide and vincristine are used alone or in combination in the compositions and methods of the invention.Therefore, compositions described herein and method can comprise more than one therapeutic agent.Suitable chemotherapeutics is at REMINGTON ' S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co.1995), with at GOODMAN ANDGILMAN ' S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, the 7th edition. (MacMillan Publishing Co.1985), and introduce in the revision version of these publications.The chemotherapeutics that other is suitable as experimental medicine, is well known to a person skilled in the art.

Toxin, as Pseudomonas exotoxin, also can be with the anti-CEA antibody of a kind of naked III class or its fragment administration.Preferably, the anti-CEA antibody of III class or its fragment are humanized MN-14 or its fragment.Other before the anti-CEA antibody of naked III class or its fragment administration, afterwards or the suitable microorganism of side by side non-coupling administration, plant or zootoxin comprise Ricin, abrin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcal enterotoxin A, Radix Phytolaccae antivirin, gelonin, diphtheria toxin, diphtherotoxin, Pseudomonas exotoxin and pseudomonas endotoxin.See, for example, Pastan etc., Cell 47:641 (1986), and Goldenberg, CA-ACancer Journal for Clinicians 44:43 (1994).Other the toxin of the present invention that is applicable to is known in those skilled in the art and at U.S. Patent number 6,077, and is open in 499, and its full content is incorporated herein by reference.These can be derived from, for example animal, plant and microbe-derived, or chemically or reorganization ground through engineering approaches design.This toxin can be the toxin of a kind of plant, microorganism or animal, or its synthetic variant.

Immunomodulator also can carry out administration with chimeric, anti-CEA antibody of III the class humanized or people of the present invention or the non-coupling of its fragment ground as cytokine.As used herein, term " immunomodifier " comprises cytokine, stem cell factor, lymphotoxin such as tumor necrosis factor (TNF), Hemopoietic factor such as interleukin are (for example, interleukin-11 (I L-1), IL-2, IL-3, IL-6, IL-10, IL-12 and IL-18), colony stimulating factor (for example, granulocyte colony-stimulating factor (GM-CSF) and CM-CSF (GM-CSF)), interferon (for example, interferon-ALPHA, β or γ), be designated as the stem cell factor of " the S1 factor ", erythropoietin and thrombopoietin.The example of suitable immunomodulator part comprises IL-2, IL-6, IL-10, IL-12, IL-18, IFN-, TNF-α etc.So the experimenter can accept a kind of naked anti-CEA antibody of III class or the cytokine of its fragment and a kind of separate administration, described cytokine can be before the anti-CEA antibody of naked III class or its segmental administration, simultaneously or carry out administration afterwards.Because some antigens also can be immunomodulators, for example, CD40 antigen, also can with the anti-CEA antibody of a kind of naked III class or its fragment administering drug combinations, before the administration of naked antibody or antibody combination, simultaneously or carry out administration afterwards.In addition, the radionuclide that is suitable for treating illing tissue includes, but not limited to ³²P, ³³P, ⁴⁷Sc, ⁵⁹Fe, ⁶⁴Cu, ⁶⁷Cu, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁰Y, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Rc, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb, ²¹²Pb and ²¹³Bi, ⁵⁸Co, ⁶⁷Ga, ^80mBr, ^99mTc, ^103mRh, ¹⁰⁹Pt, ¹¹¹In, ¹¹⁹Sb, ¹⁶¹Ho, ¹⁸⁹MOs, ¹⁹²Ir, ¹⁵²Dy, ²¹¹At, ²¹²Bi, ²²³Ra, ²¹⁹Rn, ²¹⁵Po, ²¹¹Bi, ²²⁵Ac, ²²¹Fr, ²¹⁷At, ²¹³Bi, ⁸⁸Y and ²⁵⁵Fm.Preferred radionuclide is ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁷⁷Lu and ²²⁵Ac.Also preferably, the energy of radionuclide is 20-10,000keV.

Pharmaceutically useful carrier

Naked Mus, humanization, the anti-CEA MAbs of III the class chimeric and people that is delivered to the experimenter can comprise one or more pharmaceutically useful carriers, one or more other composition, or these certain combination.

Anti-CEA antibody of non-link coupled III class of the present invention and fragment thereof can be prepared according to the known method for preparing Pharmaceutical composition.Preferably, the anti-CEA antibody of III class or its fragment are MN-14 antibody or its fragment.The phosphate buffer saline of sterilization is an example of pharmaceutically suitable carrier.Other acceptable carrier is well known in the art.See, for example, Ansel etc., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, the 5th edition (Lea ﹠amp; Febiger 1990), and Gennaro (editor), REMINGTON ' SPHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company 1990), and revised edition.

Anti-CEA antibody of non-link coupled III class of the present invention and fragment thereof can be prepared to carry out intravenous administration, described intravenous administration is undertaken by the mode of for example bolus injection or lasting infusion.Preferably, the anti-CEA antibody of III class or its fragment are MN-14 antibody or its fragment.The dosage form of injection can exist with unit dosage forms, for example, coexists as in the ampoule bottle or coexists as in the multi-agent container with the antiseptic that adds.Compositions can adopt the form of suspension, solution or Emulsion in oily or aqueous carrier, and can comprise forming agent such as suspending agent, stabilizing agent and/or dispersant.Perhaps, active component can be that powder type is to constitute a kind of suitable carriers, as the apirogen water of sterilization before use.

Can utilize other the method for pharmacy control medicament and the persistent period of naked antibody or its fragment effect.The preparation of sustained release can be compound or adsorb naked antibody and prepare by using polymer.For example, the polymer of biocompatibility comprises the substrate of the polyanhydride copolymer of the substrate of poly-(ethylene-altogether-vinylacetate) and stearic acid dimer and decanedioic acid.Sherwood etc., BiolTechnology 10:1446 (1992).From this substrate, discharge the amount of antibody or its segmental rate dependent antibody in the molecular weight of immune conjugate or antibody, substrate and the size of discrete particles.Saltzman etc., Biophys.R 55:163 (1989); Sherwood etc. see before.Other solid dosage form is at Ansel etc., PHARMACEUTICAL DOSAGEFORMS AND DRUG DELIVERY SYSTEMS, the 5th edition (Lea ﹠amp; Febiger 1990), and Gennaro (editor), REMINGTON ' S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company 1990), and introduce in the revised edition.

The anti-CEA antibody of non-link coupled III class or its fragment also can or even be passed through other parenteral route to the mammal administration in subcutaneous mode.And administration can be by continuous infusion or the bolus injection by single or multiple.Usually, give human naked antibody or its segmental dosage will according to age of patient, body weight, height, sex, general medicine situation and in the past medical history change.Usually, it is desirable giving naked antibody or its segmental dosage of the about 0.5mg/kg-20mg/kg of receiver's single venoclysis, although requirement according to circumstances can give lower or higher dosage.This dosage can carry out repetition as needs, for example 1 time every month lasting 4-10 month, preferably whenever biweekly continues for 16 weeks, and is more preferably weekly lasting 8 weeks.Can also be with frequency administration still less, as every biweekly lasting some months or with bigger frequency and/or the administration of last much longer ground.Can suitably adjust dosage and scheme by various parenteral route administrations.

For the purpose for the treatment of, treat the anti-CEA antibody of III class of effective dose or its fragment to compare the size that reduces tumor with untreated control experiment to mammal.Preferably, the anti-CEA antibody of III class or its fragment are humanized MN-14 antibody or its fragment.Be suitable for normally people of experimenter of the present invention, although also consider inhuman mammal or animal subjects.If the amount of administration is significant on the physiology, think that then the administered antibodies preparation is " a treatment effective dose ".If a kind of existence of medicament causes detectable variation on the receptor mammal physiology, so this medicament is significant on the physiology.Specifically, if the existence of antibody preparation of the present invention causes the antitumor reaction, then it is significant on the physiology.Significant effect can also be the bringing out of body fluid and/or cellular immunization in the receptor mammal on the physiology.

Although above-mentioned is particularly preferred embodiment, be appreciated that the present invention is not subjected to qualification like this.It will be understood by those skilled in the art that and can carry out suitable improvement to disclosed embodiment, this improvement is also included within the scope of the present invention, and scope of the present invention limits by claim.Be incorporated herein all references disclosed article, patent and patent application full text as a reference.

Embodiment 1: material and method

Monoclonal antibody and cell line

Having bought a kind of human thyroid myeloid cell from American type culture collection is TT.This cell monolayer grow in DMEM (Life Technologies, Gaithersburg, MD) in, described DMEM has added 10% hyclone, penicillin (100U/ml), streptomycin (100pg/ml) and L-glutaminate (2mM).Carrying out routine and going down to posterity with trypsin, 0.2%EDTA separation back pair cell.

MN-14 is the anti-CEA MAb of a kind of III class, does not react with the CEA reaction and with normal cross reacting antigen NCA and meconium antigen (Hansen etc., Cancer, 71:3478 (1993)).Here as the MN-14 and the LL2 of negative control, promptly once carried out introduction before the structure of the humanization form of anti-CD22MAb and the feature description.(Sharkey etc., CancerRes., 55:5935s (1995); Leung etc., Mol.Immunol., 32:1416 (1995)).P3x63Ag8 (MOPC-21) is from American type culture collection (Rockville, MD) a kind of incoherent mouse myeloma IgG1 of Huo Deing.By protein A purification by chromatography antibody.

Research in the body

By subcutaneous injection 2 * 10 ⁸The TT cell of individual washing female nu/nu mice (TaconicFarms, Germantown, NY) in propagation tumor, described tumor had been carried out propagation in tissue culture.Antibody is advanced to suffer from the animal of tumor via the lateral tail vein intravenous injection.The antibody amount of injection and the details of administration time are partly pointed out in the result of each research.The result provides as the gross tumor volume and the meansigma methods ± SE of individual animal.Monitor the tumor size by length, width and the degree of depth of utilizing caliper to measure tumor weekly.Gross tumor volume is calculated as the product of three kinds of measured values.Utilize Student ' s T check carrying out statistics relatively with the volume of comparison of tumor and the area under the growth curve.

Embodiment 2. sent naked hMN-14 and DTIC in 2 days behind injection TT (human thyroid marrow sample) tumor cell therapeutic alliance

In the research in front, after tumour transplatation 2 days, utilize the DTIC (the 2nd, 3 and 4 day) of 100 μ g and 25 μ g dosage and, unite with TT and give naked hMN-14 and dacarbazine at the hMN-14 of the 2nd day and the 250 μ g dosage that give weekly afterwards.The DTIC dosage of 100 μ g is united with hMN-14 than two kinds of independent treatments all more effectively (Figure 1A).But the dosage of 100 μ gDTIC produces too strong reaction, and the dosage of 25 μ g is then invalid.Surprisingly, the effect of independent MN-14 and independent DTIC is not an additivity.In other words, given independent usefulness 250 μ g hMN-14 and the result who treats with 100 μ gDTIC separately can't predict that the combination of 250 μ g hMN-14 and 100 μ gDTIC can have a kind of so significant effect.See Figure 1A.

In this research, as in the former studies, treatment starts from after the TT injection cell 2 days.The 2nd, 3,4,5,7,8,9,10,11,15 and 22 days with 100 μ g/ agent administration hMN-14, are administered once in per subsequently 7 days to reach 2.0cm up to animal dead, tumor ³Or research is because humanity is former thereby termination.The dosage of DTIC is every dose of 50 and 75 μ g/, between the dosage that it gives in research in the past.Subcutaneous injection TT cell in 60 nude mices.Injection day is a Monday, the 0th day.Referring to Figure 1B.

The result shows that the remarkable delay of tumor growth is (Figure 1B) that is caused by independent MAb treatment or independent chemotherapy.The DTIC of 75 μ g dosage in conjunction with the scheme of this hMN-14 antibody than two kinds of independent treatments all more effective significantly (p＜0.02).Unexpectedly, the DTIC of combination and MAb treatment is not an additivity.In the 7th week, to compare with 0/10 mice in untreated and MAb group with 1/10 mice in having only 75 μ g DTIC group, 8/10 mice in 75 μ g DTI C and MAb organize does not have tangible tumor.

At the 7th all mean tumour volumes is 0.018 ± 0.039cm ³(75 μ g DTIC add MN-14), 0.284 ± 0.197cm ³(75 μ g DTIC are only arranged), 0.899 ± 0.545cm ³(hMN-14 is only arranged) and 1.578 ± 0.959cm ³(untreated).Naked anti-CEA antibody and the therapeutic alliance of DTIC have strengthened independent antitumous effect with antibody or chemotherapy, and the toxicity of zero growth.The advantage of combined treatment is astonishing.

The dosage regimen general introduction: (1) the 2nd day by 11 days, except Saturday, give hMN-14 every day with 100g/ agent/mice.Antybody therapy and DTIC treatment are beginning on the same day.Gave DTIC at the 2nd, 3 and 4 day with 50 and 70 μ g/ agent, described dosage is corresponding to 5% and 7.5% MTD.The DTIC that gives a course of treatment only.

Group: 6 groups of mices, every group comprises 10 mices.

Group 1: be untreated.

Group 2. gave DTIC (Wednesday, Thursday and Friday) at the 2nd, 3 and 4 day with 50 μ g/ agent.

Group 3. gave DTIC. at the 2nd, 3 and 4 day with 75 μ g/ agent

Group 4. gave DTIC at the 2nd, 3 and 4 day with 50 μ g/ agent, added the 2nd, 3,4,5,7,8,9,10,11,15 and 22 day hMN-14 (100 μ g/ agent), was administered once up to animal dead in per subsequently 7 days, and tumor reaches 2.0cm ³Volume, or research stops.

Group 5. gave DTIC at the 2nd, 3 and 4 day with 25 μ g/ agent, added the 2nd, 3,4,5,7,8,9,10,11,15 and 22 day hMN-14 (100 μ g/ agent), was administered once up to animal dead in per subsequently 7 days, and tumor reaches 2.0cm ³Volume, or research stops.

Group 6. gave hMN-14 (determining 100 μ g/ agent) at the 2nd, 3,4,5,7,8,9,10,11,15 and 22 day, was administered once up to animal dead in per subsequently 7 days, and tumor reaches 2.0cm ³Volume, or research stops.

The survival of monitoring animal.Measure tumor and body weight weekly.

Method: at second day, with 2, the DTIC of 200mg/ phial was reconstructed with the 19.7ml aquesterilisa and is used for injection.The solution that obtains comprises the dacarbazine of 10mg/ml, and the pH scope is 3.0-4.0.Utilize this solution to carry out dilution described below as required and residue is carried out the use subsequently of freezing do with the aliquot of 1ml.

Group 2 and 4: the solution of preparation 5ml 0.5mg/ml.The 0.5mg/ml/ mice solution of intravenous injection 100 μ l.

Group 3 and 5: the solution of preparation 5ml 0.75mg/ml.The 0.75mg/ml/ mice solution of intravenous injection 100 μ l.

Estimate the amount of hMN-14.In the mice of 1mg/ml hMN-14 peritoneal injection in group 4,5 and 6 with 100 μ l.

The radioimmunoassay therapy studies of embodiment 3. in people MTC heteroplastic transplantation model

The applicant has developed a kind of people MTC allotransplant that utilizes the people MTC cell line of the called after TT that produces CEA and calcitonin, uses radiolabeled anti-CEA MAbs to carry out model ([Stein, the 1999﹠amp of the experiment radioimmunoassay therapy of MTC; Num; 82], see appendix).By subcutaneous vaccination 2 * 10 ⁸Individual cell is set up the MTC tumor and is allowed its growth 2-5 week before injection MAbs in nude mice.Carry out bio distribution and RAIT research with MN-14 subsequently, it shows and the TT cell effect for the flow cytometry method.In these researchs, all utilize Ag8 and Mu-9 as negative control MAbs.Utilize the preliminary study of the less tumor of about 0.08g to show injection ¹³¹I-MN-14 7 days afterwards is with the common injection of having only 12.6%ID/g ¹²⁵The I-Ag8 contrast is compared, and the percentage ratio (%ID/g) of injected dose/gram tumor is 68.9%.Use bigger tumor (five week of growth in nude mice; Average tumor weight=0.404g), in injection ¹²⁵The ID/g% of 7 days observed tumors is 12.4% after the I-MN-14.But, inject altogether ⁸⁸The ID/g% of Y-MN-14 is 50.5%, or ratio ¹²⁵I-MN-14 is high 4.1 times.With ⁸⁸The ratio of the tumor of Y-MN-14 and blood, lung, liver, spleen and kidney is also than usefulness ¹²⁵I-MN-14's is higher, and equates with the ratio of bone with the tumor of these two kinds of medicaments.Work as utilization ¹²⁵I-MN-14 and ⁸⁸Y-MN-14 bio distribution data are predicted usefulness respectively ¹³¹I-MN-14 and ⁹⁰During the tumour radiotherapy dosage of Y-MN-14, ⁹⁰The radiation absorbed dose of sending on the MTD of Y-MN-14 (150 μ Ci) ratio exists ¹³¹The radiation absorbed dose of sending on the MTD of I-MN-14 (275 μ Ci) high 1.75 times (4900cGy is to 2800cGy).

Treatment in this model is determined ⁹⁰Y-MN-14 is a ratio ¹³¹The better therapeutic agent of I-MN-14.In 5 all big tumors, with usefulness ¹³¹I-MN-14 only has tumor growth delay to compare ⁹⁰The tumor growth of seeing for 5 weeks on the MTD of Y-MN-14 suppresses (Fig. 2) fully.And, when less 2 weeks of treatment during big tumor, ⁹⁰See average 60% gross tumor volume on the MTD of Y-MN-14 and reduced, be accompanied by some tumor regressions completely.Compare with the relative tumor growth fast in not treating animal or those MTD that treats with contrast MAbs, these Graft Versus Tumor are very significant.Thereby our preclinical study shows that this zootype is to be suitable for very much the experimental RAIT's that carries out with anti-CEA MAbs.

With ¹³¹I compares ⁹⁰Path and higher-energy that Y is long add ⁹⁰Y is kept the fact of long period to cause being delivered to the radiological dose of tumor raising and thereby having caused waiting effective more curative effect under the toxicity by target cell.If our using is remaining ¹³¹The result of I (refs) can be generalized to the MN-14 among the MTC, and we can expect remnants so ¹³¹In the size tumor that I here studies with ⁹⁰Y is effectively same at least, and has advantage probably in disposing the micrometastasis disease or as postoperative auxiliary treatment.

Embodiment 4: chemotherapy

To four kinds of medicines, amycin, DTIC (dacarbazine), cyclophosphamide and vincristine are assessed the effect of their TT MTC xenograft growths in nude mice individually or in combination.Based on every kind of medicine clinically with mg/m ²For the basis dosage of people's administration being selected dosage.Tumor size and body weight are measured in the survival of monitoring animal weekly.Fig. 3 shows the tumor growth curve of animal in this research.By individually administration, amycin, DTIC and cyclophosphamide have produced significant growth inhibited, and vincristine does not produce significant growth inhibited, although the growth delay that is caused by DTIC significantly longer than other medicines.For every group of time on a rough average that doubles be: untreated, 1 week; Amycin, 2.5 weeks; DTIC, 7.5 weeks; Cyclophosphamide, 3 weeks; And vincristine, 1.5 weeks.The combination of amycin and DTIC is compared with two kinds of independent medicines and has been improved effectiveness, will increase to for 10 weeks the average time that double.But, to compare with independent DTIC, the effectiveness of the raising of amycin and DTIC combination does not reach 95% confidence level.AUC P value relatively is as follows: amycin+DTIC is to amycin, P＜0.01, and amycin+DTIC is to DTIC, P＜0.1.The therapy of 4 kinds of medicines extended to for 12 weeks with the average time that doubles; For the two comparison P＜0.01 of amycin and DTIC.

Individual drug has only shown significant difference to DTIC and cyclophosphamide to the logarithm rank analysis of the survival data of untreated fish group.Respectively with DTIC and cyclophosphamide treatment group 11 the week compare with 8 weeks, the mean survival time of untreated control group was 4 weeks, and drug regimen is greater than 12 weeks.As by the measured toxicity of body weight loss for all seminar all within acceptable scope.After with 1 week of the treatment in the mice of all 4 kinds of drug treating, observe the maximum weight loss, body weight loss is between the 3-12% scope.

Embodiment 5. associating radioimmunotherapy and chemotherapy are carried out the treatment of MTC

RAIT adds 4 kinds of drug regimens

By the TT growth in untreated mice is compared with the mice of 4 kinds of medicines of above-mentioned use (amycin, DTIC, cyclophosphamide and vincristine) scheme treatment, assess using ⁹⁰The RAIT that the anti-CEA MAb of Y MN-14 carries out and 4 kinds of effects that drug regimen combines, the 50%MTD of 100% maximum tolerated dose (MTD) of RAIT (105 μ Ci), RAIT, and the 50%MTD of RAIT combination is with those of 4 kinds of drug treating.Fig. 4 is presented at TT growth of tumor curve in the mice that gives various therapeutic schemes.Compare with untreated animal, all four treatment groups have all produced significant raising on rendeing a service.In view of the average doubling time of not treating in the animal roughly was 1.5 weeks, the chemotherapy of carrying out with 4 kinds of medicines the average doubling time was extended to for 10 weeks and independent RAI T respectively at 50% with drawn during 100%MTD doubling time in 4 weeks and 8 weeks.As expected, 100%RAIT group and 4 kinds of therapeutic scheme groups the two all organize better significantly than 50%RAIT.The most important thing is, compare, 50%RAIT and 4 kinds of pharmaceutical admixtures are combined improved the result, further the average doubling time was extended to for 12.5 weeks with two kinds of therapies.For the comparison of therapeutic alliance and 4 kinds of pharmaceutical admixtures, P＜0.02, for the comparison of 100%RAIT, P＜0.01.

The average weight loss (nadir) in treatment 1 week of back is 9% for 100%RAIT and 4 kinds of pharmaceutical admixtures, but adds that for the 50%RAIT of combination 4 kinds of pharmaceutical admixtures are 15%.In addition, in the combination treatment group, animal died from after 3 weeks of treatment and second animal has the loss in weight greater than 20%.Thereby this treatment has exceeded maximum tolerated dose.

RAIT adds the chemotherapy of 2 kinds of pharmaceutical admixtures

Also in this MTC xenograft models, assessment is using ⁹⁰The RAIT that the anti-CEA MAb of Y MN-14 carries out and 2 kinds of effects that drug regimen combines, described 2 kinds of drug regimens are made up of amycin and DTIC.The roughly doubling time of each group is: untreated, and 1.5 weeks; Amycin adds DTIC, 8 weeks; The MTD of RAIT, 10 weeks; In conjunction with MTD, greater than 12 weeks with the RAIT of 2 kinds of pharmaceutical admixtures of 25-75%.Thereby independent RAIT is more effective than 2 kinds of pharmaceutical admixtures, and the most significantly, and RAIT and 2 kinds of pharmaceutical admixtures are combined to compare with any independent therapy has produced the result who improves.For the comparison of combined treatment and 2 kinds of pharmaceutical admixtures, P＜0.005, for the comparison of independent RAIT, P＜0.02.

The average weight loss in treatment back (nadir) 1-2 week is 2-8% for each group, observes when the 2nd week 13% the loss in weight except 100%RAIT adds 2 kinds of medicine chemotherapy group of 75%.In addition, in this therapeutic alliance group, two animals die from treatment back 3-4 week and a loss in weight that has experienced greater than 20%.Thereby the amycin and the DTIC that add 75% dosage level in the treatment of 100%RAIT have exceeded MTD, and 50% and the 100%RAIT combination of these 2 kinds of drug regimens then can be tolerated.

RAIT adds amycin

Because in the past publication had been reported combination (Stein etc., Clin CancerRes., the 5:3199s (1999) of RAIT and amycin in this model; Behr etc., Cancer Res.57:5309 (1997)), can add the amycin scheme to RAIT and do directly relatively.Also RAIT being added 4 kinds of pharmaceutical admixtures does directly relatively.Compare with untreated animal, all treatments have all produced significant effectiveness.The average doubling time that RAIT adds amycin was 12 weeks.In this research, the amycin of the full MTD of RAIT and 50% and DTIC or 4 kinds of pharmaceutical admixtures are combined extended to for 15 weeks with the average doubling time, did not have the significant difference on the statistics between these two groups.In these researchs, observed the basic number of objective reaction.Add the amycin treatment with RAIT after, 3 animal complete reactions are arranged, 2 animal part reactions, and in whole 10 mices, 5 at least 4 weeks of animal stable disease.RAIT adds 2 kinds of pharmaceutical admixtures and objective reaction is increased to 10 of 12 animals has only complete reaction and 2 to have only partial reaction, and RAIT adds 7 mice complete reactions and 2 partial reactions that 4 kinds of therapeutic schemes cause 9 mices.

RAIT adds DTIC

Because DTIC is the most effective chemotherapeutics when individually dosed, so assess the effectiveness that RAIT adds DTIC by the effectiveness that adds amycin and DTIC with RAIT.It will be important for clinical practice that amycin is left out from therapeutic scheme, so that avoid the additional toxicity of this medicine, and particularly known cardiac toxicity.As shown in Figure 5, two seminar that accept chemotherapy combined RAIT, amycin and DTIC or have only DTIC are equal to each other haply, and the two is all more effective than the treatment of independent form.AUC P value relatively is as follows: RAIT+DTIC is to DTIC, P＜0.01, and RAIT+DTIC is to RAIT, P＜0.05.Compare with 9 weeks with RAIT7.5 week with independent DTIC respectively, RAIT adds the average doubling time that DTIC and RAIT add amycin and DTIC group and was respectively for 15.5 all and 14 weeks.Thereby the treatment that RAIT adds the cooperative programs of DTIC has prolonged 100% with the average doubling time on the DTIC chemotherapy.Adding DTIC and RAIT by AUC or Time-Series analysis at RAIT adds between amycin and the DTIC group and does not observe significant difference.

Embodiment 6: the research of carrying out separately with naked anti-CEA

The treatment of carrying out with naked hMN-14

In order to study the influence of unlabelled hMN-14 for TT tumor growth in nude mice, after tumor cell injection one day or 11 days is by intravenous administration single injection hMN-14.Fig. 6 demonstration is compared with untreated contrast, uses the tumor growth curve of the animal of 0.5mg hMN-14/ mice processing.Untreated fish group comprises 16 animals; Two processed group comprise 10 animals for every group.Between 1 day processed group behind untreated fish group and the tumor injection, observe significant growth delay.Observed the significant difference (p＜0.05) of average tumor size from the 32nd day to 93 days.Compare with the animal of being untreated, the inhibition of the 64-70% of tumor size was arranged between the 32nd day and 60 days in the MN-14 processed group.Between the 11st day group and the average tumor size of untreated fish group, there is not significant difference.Also see the remarkable delay of tumor growth by the t check analysis under growth curve.For the untreated fish group of comparing with the group handled in 1 day behind the tumor injection, P＜0.05, but really not so the group that 11 days handle behind tumor injection.

The specificity of treatment

Fig. 7 has summed up the result for the The specificity of antitumor reaction.Unlabelled hMN-14 in the nude mice is compared with negative control humanization MAb, hLL2 (anti-CD22) and the effect of Mus MN-14 for the effect of TT tumor growth.Administration in a day (intravenous ground) MAbs (0.5mg/ mice) after the TT cell gives the dosage of three weekly 0.5mg/ mices subsequently.The group of 15 animals of research.Observedly in first research in this research, obtain affirmation because of growth inhibited with 0.5mghMN-14 treatment.Since the 23rd day, observe the significant difference (p＜0.05) of average tumor size between hMN-14 and untreated fish group.Mean tumour volume in the group of handling with hMN-14 in the 37th day is 42.7% of a untreated control animal.The processing of carrying out with Mus MN-14 produces the result similar to hMN-14.The processing of carrying out with hLL2 does not slow down tumor growth; Opposite have little (inapparent) to increase on growth rate.For example, compare with 29% of hLL2 processed group with untreated 40%, in the 37th day 87% the tumor of handling with hMN-14 less than 0.5cm ³To being presented at untreated fish group in the T of the following area of growth curve check analysis and, still but not having significant difference with the group that hLL2 handles with the significant difference (p＜0.05) between hMN-14 or the Mus MN-14 processed group.In addition, the hMN-14 group significantly is different from the hLL2 group, but but different significantly with the animal of Mus MN-14 processing.

The effect of dosage

For the influence of the dosage of studying unlabelled hMN-14, the hMN-14 dosage that increases is gradually assessed for TT tumor growth in the nude mice.Give a plurality of antibody dosages after 1 day at the TT cell, stop up to research once in a week subsequently.In the group of six mices weekly dosage at 0.125mg in the scope of 2.0mg hMN-14/ mice.Between untreated fish group and all processed group, observe the significant difference (Fig. 8) of average tumor size and the following area of growth curve.For example, the mean tumour volume in two minimum hMN-14 processed group is the 27-40% of tumor size in being untreated animal between the 21st day and the 49th day.As if more effective with the processing of carrying out than low dosage 0.125mg and 0.25mg than the processing of carrying out with higher dosage, but difference does not reach the significance on the statistics.

Timing

By the administration date that changes MAb assess time between the initial dose of TT injection and hMN-14 for nude mice in the influence of TT tumor growth.1,3 or 7 day administration hMN-14 (0.25mg) after the TT cell finishes up to research subsequently once in a week.The group of 7-8 animal of research.The results are summarized among Fig. 9.Between untreated fish group and all three processed group, observe the significant difference (p＜0.05) of average tumor size.But the difference of average tumor size is only at a time point between untreated mice and the 7th day processed group, and the 28th day is significant.The mice of handling in the 1st day produced significant difference at 21-77 days, and the mice of handling in the 3rd day produced significant difference at 21-70 days.The T check analysis of the area under the growth curve is shown the significant growth inhibited of comparing with untreated fish group for 1 day or the group demonstration handled with hMN-14 in 3 days the administration of TT cell after.This analysis is for not reaching 95% confidence limit (at the 5th all p=0.057) in untreated fish group and the difference between the group of processing in the 7th day.

Embodiment 7: the naked anti-CEA of combination adds the treatment of DTIC to MTC

For study the effectiveness whether naked hMN-14 can strengthen DTI C, grow the TT cell is arranged nude mouse with DTIC (75 μ g/ agent), unite the course of treatment with one section unlabelled MAb.Behind subcutaneous injection TT cell 2 days the beginning, for three days on end with 75 μ g/ agent administration DTI C as a course of treatment.HMN-14MAb treatment starts from the same day with first dose of DTIC, at first two weeks with lasting 5 days of 100 μ g/ agent/skies, weekly twice subsequently.These MAb therapies or independent chemotherapy cause the remarkable delay (Figure 10) of tumor growth.The DTIC of 75 μ g dosage combination with the hMN-14 of this programme than two kinds of independent treatments effective (P＜0.02) more significantly.After 7 weeks, with in 75 μ gDTIC group is only arranged 1/10 with in untreated fish group with have only 0/10 in the MAb group to compare, 8/10 mice in 75 μ g DTIC+MAb organize does not have palp tumor.Mean tumour volume during the 7th week is 0.018+0.039cm ³(75 μ gDTIC+hMN-14), 0.284+0.197cm ³(75 μ g DTIC), 0.899+0.545cm ³(hMN-14) and 1.578+0.959cm ³(untreated).

Anti-CEA MAb MN-14 has shown unexpected antitumor effectiveness in MTC, and needn't be coupled on a kind of cytotoxic agent.Beginning in 3 weeks and continuing 2 months at least, in hMN-14 difference that handle and that observe the average tumor size between untreated group.The processing of carrying out with the negative control MAbs of the isotype coupling growth of tumor that do not slow down.This is first evidence that suppresses tumor with " naked " anti-CEA MAb.But naked anti-CEA MAb and the therapeutic alliance of DTIC have strengthened the independent antibody or the antitumous effect of chemotherapy.The advantage support of cooperative programs treatment incorporates in the chemotherapy regimen CEA-MAb therapy to carry out MTC control into.The tumor cytotoxicity mechanism of MN-14 is unknown, may relate to some mechanism.The research of Ti Chuing herein will provide the basis of understanding these observed results and the basis of supporting clinical trial.

Although above-mentioned is particularly preferred embodiment, be appreciated that the present invention is not subjected to qualification like this.It will be understood by those skilled in the art that and can carry out suitable improvement to disclosed embodiment, this improvement is also included within the scope of the present invention, and scope of the present invention limits by claim.

Be incorporated herein all references disclosed article, patent and patent application full text as a reference.

Claims (68)

1.III the anti-CEA monoclonal antibody of class or its Fab and at least a therapeutic agent are used for the treatment of application in the medicine of cancer in preparation, the anti-CEA monoclonal antibody of wherein said III class or its Fab not with described therapeutic agent coupling, and the described combination of wherein anti-CEA Mab and therapeutic agent is for the treatment human cancer, than the effect of the anti-CEA Mab of independent anti-CEA Mab or independent therapeutic agent or administration respectively and therapeutic agent add and, all more effective.

2. the application of claim 1, the anti-CEA MAb of wherein said III class or its Fab are humanized, wherein said humanized MAb has kept the anti-CEA binding specificity of III class of the anti-CEA MAb of Mus III class basically.

3. the application of claim 1, the anti-CEA MAb of wherein said III class or its Fab are chimeric MAb, wherein said chimeric MAb has kept the anti-CEA binding specificity of III class of the anti-CEA MAb of Mus III class basically.

4. the application of claim 1, the anti-CEA monoclonal antibody of wherein said III class or its Fab are MN-14 antibody or its Fab.

5. the application of claim 1, wherein said MN-14 monoclonal antibody or its Fab comprise the complementary determining region (CDRs) of Mus MN-14 monoclonal antibody, and the CDRs of the variable region of light chain of wherein said MN-14 antibody comprises the CDR1 that contains aminoacid sequence KASQDVGTSVA; The CDR2 that contains aminoacid sequence WTSTRHT; With the CDR3 that contains aminoacid sequence QQYSLYRS; And the CDRs of the variable region of heavy chain of the anti-CEA antibody of described III class comprises the CDR1 that contains TYWMS; The CDR2 that contains EIHPDSSTINYAPSLKD; With the CDR3 that contains LYFGFPWFAY.

6. the application of claim 5, wherein said MN-14 monoclonal antibody and CEA reaction, but do not react with normal cross-reactive antigen (NCA) and meconium antigen (MA).

7. the application of claim 6, wherein said MN-14 monoclonal antibody or its Fab are humanized MN-14 antibody or its Fab.

8. the application of claim 6, wherein said MN-14 monoclonal antibody or its Fab are chimeric MN-14 antibody or its Fabs.

9. the application of claim 6, wherein said MN-14 monoclonal antibody or its Fab are people MN-14 antibody or its Fab completely.

10. the application of claim 7, the light chain of wherein said humanization MN-14 antibody or its Fab and the framework region of variable region of heavy chain (FRs) comprise the aminoacid of at least one replacement from the corresponding FRs of Mus MN-14 monoclonal antibody.

11. the application of claim 10, wherein said humanization MN-14 antibody or its Fab comprise at least one aminoacid from the described corresponding FR of described Mus MN-14 antibody, and described aminoacid is selected from the V of Mus variable region of heavy chain KLHuVhAIGA or hMN-14 or hMN14 _HAmino acid residue 24,28,30,48,49,74 and 94.

12. the application of claim 10, wherein said humanization MN-14 antibody or its Fab comprise at least one aminoacid from the described corresponding FR of described Mus MN-14 variable region of light chain.

13. the application of claim 10, wherein said humanization MN-14 antibody or its Fab comprise the variable region of light chain of MN-14 V κ, REIV κ or hMN14 V κ, and KLHuVhAIGA's or hMN-14 V κ's variable region of heavy chain.

14. any one application of claim 1-13, wherein said Fab are selected from F (ab ') ₂, Fab ', Fab, Fv and sFv.

15. any one application of claim 1-13, that wherein said therapeutic agent is selected from is humanized, chimeric, the people's or Mus monoclonal antibody or its Fab, described monoclonal antibody or its Fab are selected from the anti-CEA monoclonal antibody of II class, the anti-CEA monoclonal antibody of III class and Fab thereof, and side by side or according to priority carry out administration with the treatment effective dose.

16. the application of claim 15, wherein said antibody or its Fab are naked or are coupled to antibody on the another kind of therapeutic agent.

17. any one application of claim 1-13, wherein said therapeutic agent is selected from immune conjugate, hormone or its combination of naked antibody, cytotoxic agent, medicine, radionuclide, immunomodulator, photosensitive therapeutic agent, CEA or non-CEA antibody, optional being formulated in a kind of pharmaceutically useful carrier.

18. the application of claim 17, wherein said therapeutic agent is selected from and EGP-1, EGP-2, MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, EGP-2, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn, Thomson-Friedenreich antigen, neoplasm necrosis antigen, VEGF or other tumor vessel generation antigen, Ga733 and composite reaction thereof humanized, chimeric, the people's or Mus monoclonal antibody or its Fab, and side by side or according to priority described experimenter is carried out administration with the treatment effective dose.

19. the application of claim 18, wherein said antibody or its Fab are naked or are coupled on the another kind of therapeutic agent.

20. any one application of claim 1-13, wherein said therapeutic agent are not DTIC.

21. the application of claim 17, wherein said cytotoxic agent are a kind of medicine or toxin.

22. the application of claim 21, wherein said medicine has the pharmacological characteristics that is selected from antimitotic agent, alkylating agent, antimetabolite, anti-angiogenic agent, apoptosis agent, alkaloid, COX-2 and antibiotic and combination thereof.

23. the application of claim 21, wherein said medicine are selected from urea, methylhydrazine derivant, adrenal cortex inhibitor, antagonist, endostatin, paclitaxel, camptothecine, amycin and the combination thereof of chlormethine, aziridine derivative, alkyl sulfonic ester, nitroso ureas, triazenes, folic acid, anthracycline, taxane, cox 2 inhibitor, pyrimidine, purine, antimetabolite, antibiotic, enzyme, epipodophyllotoxin, iridium-platinum complex, vinca alkaloids, replacement.

24. the application of claim 21, wherein said toxin is the toxin of microorganism, plant or animal, is selected from Ricin, abrin, alpha toxin, ZAOCAO toxin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcal enterotoxin A, Radix Phytolaccae antivirin, gelonin, diphtheria toxin, diphtherotoxin, Pseudomonas exotoxin and pseudomonas endotoxin.

25. the application of claim 17, wherein said immunomodulator are selected from cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), stem cell factor, erythropoietin, thrombopoietin and combination thereof.

26. the application of claim 25, wherein said lymphotoxin is tumor necrosis factor (TNF), described Hemopoietic factor is interleukin (IL), described colony stimulating factor is granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimutaing factor (GM-CSF), described interferon is interferon-ALPHA, β or γ, and described stem cell factor is designated as " the S1 factor ".

27. the application of claim 25, wherein said immunomodulator comprise IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, IFN-, TNF-α or its combination.

28. the application of claim 17, the energy of wherein said radionuclide are 20-10,000keV.

29. the application of claim 28, wherein said radionuclide is selected from ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁸Y, ²²⁵Ac, ¹⁷⁷Lu, ¹⁸⁸Re, ¹⁸⁶Re and combination thereof.

30. the application of claim 17, wherein said photosensitive therapeutic agent is a kind of chromogen or dyestuff.

31. the application of claim 22, wherein said alkylating agent are dacarbazine.

32. the application of claim 18, wherein said antibody is 17-1A.

33. the purposes of each of claim 1-13, wherein said cancer are to express the cancer of carcinoembryonic antigen.

34. the purposes of each of claim 1-13, wherein said cancer is a colorectal carcinoma.

35.III the anti-CEA antibody of class or its Fab and at least a therapeutic agent are used for the treatment of application in the medicine of cancer in preparation, the described combination of wherein anti-CEA Mab and therapeutic agent is for the treatment human cancer, than the effect of the anti-CEA Mab of independent anti-CEA Mab or independent therapeutic agent or administration respectively and therapeutic agent add and, all more effective.

36. the application of claim 35, the anti-CEA MAb of wherein said III class or its Fab are humanized, and wherein said humanized MAb has kept the anti-CEA binding specificity of III class of the anti-CEA MAb of Mus III class basically.

37. the application of claim 35, the anti-CEA MAb of wherein said III class or its Fab are chimeric MAb, and wherein said chimeric MAb has kept the anti-CEA binding specificity of III class of the anti-CEA MAb of Mus III class basically.

38. the application of claim 35, the anti-CEA monoclonal antibody of wherein said III class or its Fab are MN-14 antibody or its Fab.

39. the application of claim 38, wherein said MN-14 monoclonal antibody or its Fab comprise the complementary determining region (CDRs) of Mus MN-14 monoclonal antibody, and the CDRs of the variable region of light chain of wherein said MN-14 antibody comprises the CDR1 that contains aminoacid sequence KASQDVGTSVA; The CDR2 that contains aminoacid sequence WTSTRHT; With the CDR3 that contains aminoacid sequence QQYSLYRS; And the CDRs of the variable region of heavy chain of the anti-CEA antibody of described III class comprises the CDR1 that contains TYWMS; The CDR2 that contains EIHPDSSTINYAPSLKD; With the CDR3 that contains LYFGFPWFAY.

40. the application of claim 39, wherein said MN-14 monoclonal antibody and CEA reaction, but do not react with normal cross-reactive antigen (NCA) and meconium antigen (MA).

41. the application of claim 40, wherein said MN-14 monoclonal antibody or its Fab are humanized MN-14 antibody or its Fab.

42. the application of claim 40, wherein said MN-14 monoclonal antibody or its Fab are chimeric MN-14 antibody or its Fabs.

43. the application of claim 40, wherein said MN-14 monoclonal antibody or its Fab are people MN-14 antibody or its Fab completely.

44. the application of claim 41, the light chain of wherein said humanization MN-14 antibody or its Fab and the framework region of variable region of heavy chain (FRs) comprise the aminoacid of at least one replacement from the corresponding FRs of Mus MN-14 monoclonal antibody.

45. the application of claim 44, wherein said humanization MN-14 antibody or its Fab comprise at least one aminoacid from the described corresponding FR of described Mus MN-14 antibody, and described aminoacid is selected from the V of Mus variable region of heavy chain KLHuVhAIGA or hMn-14 or hMN14 _HAmino acid residue 24,28,30,48,49,74 and 94.

46. the application of claim 44, wherein said humanization MN-14 antibody or its Fab comprise at least one aminoacid from the described corresponding FR of described Mus MN-14 variable region of light chain.

47. the application of claim 44, wherein said humanization MN-14 antibody or its Fab comprise the variable region of light chain of MN-14 V κ, REIV κ or hMN14 V κ, and KLHuVhAIGA's or hMN-14 V κ's variable region of heavy chain.

48. any one application of claim 35-47, wherein said Fab are selected from F (ab ') ₂, Fab ', Fab, Fv and sFv.

49. any one application of claim 35-47, that wherein said therapeutic agent is selected from is humanized, chimeric, the people's or Mus monoclonal antibody or its Fab, described monoclonal antibody or its Fab are selected from the anti-CEA monoclonal antibody of II class, the anti-CEA monoclonal antibody of III class and Fab thereof, and side by side or according to priority carry out administration with the treatment effective dose.

50. the application of claim 49, wherein said antibody or its Fab are naked or are coupled to antibody on the another kind of therapeutic agent.

51. any one application of claim 35-47, wherein said therapeutic agent is selected from immune conjugate, hormone or its combination of naked antibody, cytotoxic agent, medicine, radionuclide, immunomodulator, photosensitive therapeutic agent, CEA or non-CEA antibody, optional being formulated in a kind of pharmaceutically useful carrier.

52. the application of claim 51, wherein said therapeutic agent is selected from and EGP-1, EGP-2, MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, EGP-2, HER2/neu, BrE3, Le-Y, A3, A33, Ep-CAM, AFP, Tn, Thomson-Friedenreich antigen, neoplasm necrosis antigen, VEGF or other tumor vessel generation antigen, Ga733 and composite reaction thereof humanized, chimeric, the people's or Mus monoclonal antibody or its Fab, and side by side or according to priority described experimenter is carried out administration with the treatment effective dose.

53. the application of claim 52, wherein said antibody or its Fab are naked or are coupled on the another kind of therapeutic agent.

54. any one application of claim 35-47, wherein said therapeutic agent are not DTIC.

55. the application of claim 51, wherein said cytotoxic agent are a kind of medicine or toxin.

56. the application of claim 51, wherein said medicine has the pharmacological characteristics that is selected from antimitotic agent, alkylating agent, antimetabolite, anti-angiogenic agent, apoptosis agent, alkaloid, COX-2 and antibiotic and combination thereof.

57. the application of claim 55, wherein said medicine are selected from urea, methylhydrazine derivant, adrenal cortex inhibitor, antagonist, endostatin, paclitaxel, camptothecine, amycin and the combination thereof of chlormethine, aziridine derivative, alkyl sulfonic ester, nitroso ureas, triazenes, folic acid, anthracycline, taxane, cox 2 inhibitor, pyrimidine, purine, antimetabolite, antibiotic, enzyme, epipodophyllotoxin, iridium-platinum complex, vinca alkaloids, replacement.

58. the application of claim 55, wherein said toxin is the toxin of microorganism, plant or animal, is selected from Ricin, abrin, alpha toxin, ZAOCAO toxin, ribonuclease (RNA enzyme), DNA enzyme I, staphylococcal enterotoxin A, Radix Phytolaccae antivirin, gelonin, diphtheria toxin, diphtherotoxin, Pseudomonas exotoxin and pseudomonas endotoxin.

59. the application of claim 51, wherein said immunomodulator are selected from cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), stem cell factor, erythropoietin, thrombopoietin and combination thereof.

60. the application of claim 59, wherein said lymphotoxin is tumor necrosis factor (TNF), described Hemopoietic factor is interleukin (IL), described colony stimulating factor is granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimutaing factor (GM-CSF), described interferon is interferon-ALPHA, β or γ, and described stem cell factor is designated as " the S1 factor ".

61. the application of claim 51, wherein said immunomodulator comprise IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, IFN-, TNF-α or its combination.

62. the application of claim 51, the energy of wherein said radionuclide are 20-10,000keV.

63. the application of claim 62, wherein said radionuclide is selected from ¹²⁵I, ¹³¹I, ⁹⁰Y, ⁸⁸Y, ²²⁵Ac, ¹⁷⁷Lu, ¹⁸⁸Re, ¹⁸⁶Re and combination thereof.

64. the application of claim 51, wherein said photosensitive therapeutic agent is a kind of chromogen or dyestuff.

65. the application of claim 56, wherein said alkylating agent are dacarbazine.

66. the application of claim 52, wherein said antibody is 17-1A.

67. the purposes of each of claim 35-47, wherein said cancer are to express the cancer of carcinoembryonic antigen.

68. the purposes of each of claim 35-47, wherein said cancer is a colorectal carcinoma.

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